GH61 Polypeptide Variants and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to GH61 polypeptide variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/358,642 filed May 15, 2014, which is a 35 U.S.C. § 371 nationalapplication of PCT/US2012/066278 filed Nov. 21, 2012, which claimspriority or the benefit under 35 U.S.C. § 119 of U.S. ProvisionalApplication No. 61/562,277 filed Nov. 21, 2011, the contents of whichare fully incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to GH61 polypeptide variants,polynucleotides encoding the variants, methods of producing thevariants, and methods of using the variants.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars can easily befermented by yeast into ethanol.

WO 2005/074647, WO 2008/148131, and WO 2011/035027 disclose isolatedGH61 polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 and WO 2012/149344 disclose isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Trichoderma reesei. WO 2009/085935, WO2009/085859, WO 2009/085864, and WO 2009/085868 disclose isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Myceliophthora thermophila. WO 2010/138754discloses an isolated GH61 polypeptide having cellulolytic enhancingactivity and the polynucleotide thereof from Aspergillus fumigatus. WO2011/005867 discloses an isolated GH61 polypeptide having cellulolyticenhancing activity and the polynucleotide thereof from Penicilliumpinophilum. WO 2011/039319 discloses an isolated GH61 polypeptide havingcellulolytic enhancing activity and the polynucleotide thereof fromThermoascus sp. WO 2011/041397 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Penicillium sp. (emersonii). WO 2011/041504 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thermoascus crustaceus. WO 2012/030799discloses GH61 polypeptides having cellulolytic enhancing activity andthe polynucleotides thereof from Aspergillus aculeatus. WO 2012/113340discloses GH61 polypeptides having cellulolytic enhancing activity andthe polynucleotides thereof from Thermomyces lanuginosus. WO 2012/146171discloses GH61 polypeptides having cellulolytic enhancing activity andthe polynucleotides thereof from Humicola insolens. WO 2008/151043discloses methods of increasing the activity of a GH61 polypeptidehaving cellulolytic enhancing activity by adding a soluble activatingdivalent metal cation to a composition comprising the polypeptide.

WO 2012/044835 and WO 2012/044836 disclose GH61 polypeptide variantshaving cellulolytic enhancing activity with improved thermal activityand thermostability.

The present invention provides GH61 polypeptide variants with increasedthermostability.

SUMMARY OF THE INVENTION

The present invention relates to isolated GH61 polypeptide variants,comprising a substitution at one or more (e.g., several) positionscorresponding to positions 105, 154, 188, 189, 216, and 229 of themature polypeptide of SEQ ID NO: 30, wherein the variants havecellulolytic enhancing activity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to processes for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a GH61polypeptide variant of the present invention. In one aspect, theprocesses further comprise recovering the degraded or convertedcellulosic material.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a GH61polypeptide variant of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a GH61 polypeptide variant of the present invention. In oneaspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the processes further compriserecovering the fermentation product from the fermentation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence (SEQ ID NO: 29) and the deducedamino acid sequence (SEQ ID NO: 30) of an Aspergillus fumigatus geneencoding a GH61B polypeptide having cellulolytic enhancing activity.

FIG. 2 shows a restriction map of plasmid pMMar44.

FIG. 3 shows a restriction map of plasmid pMMar49.

FIG. 4 shows a restriction map of plasmid pMMar45.

FIG. 5 shows a restriction map of plasmid pDFng113.

FIG. 6 shows the effect of addition of Aspergillus fumigatus GH61Bpolypeptide variants in the conversion of PCS by a high-temperaturecellulase composition at 50° C., 55° C., and 60° C.

FIG. 7 shows a restriction map of plasmid pDFng153-4.

FIG. 8 shows a restriction map of plasmid pDFng154-17.

FIG. 9 shows a restriction map of plasmid pDFng155-33.

FIG. 10 shows a restriction map of plasmid pBGMH16.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Crystalline cellulose degradation: New insight intothe function of cellobiohydrolases, Trends in Biotechnology 15: 160-167;Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why soefficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178).Cellobiohydrolase activity is determined according to the proceduresdescribed by Lever et al., 1972, Anal. Biochem. 47: 273-279; vanTilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh andClaeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988,Eur. J. Biochem. 170: 575-581.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperaturesuch as 40° C.−80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C.,and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0,compared to a control hydrolysis without addition of cellulolytic enzymeprotein. Typical conditions are 1 ml reactions, washed or unwashedpretreated corn stover (PCS), 5% insoluble solids, 50 mM sodium acetatepH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugar analysis byAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA). 40° C.−80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of the mature polypeptide thereof, wherein the fragment hascellulolytic enhancing activity. In one aspect, a fragment contains atleast 85% of the amino acid residues, e.g., at least 90% of the aminoacid residues or at least 95% of the amino acid residues of the maturepolypeptide of a GH61 polypeptide.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchan improved property includes, but is not limited to, increasedthermostability.

Increased thermostability: The term “increased thermostability” means ahigher retention of cellulolytic enhancing activity of a GH61polypeptide variant after a period of incubation at a temperaturerelative to the parent. The increased thermostability of the variantrelative to the parent can be assessed, for example, under conditions ofone or more (e.g., several) temperatures. For example, the one or more(e.g., several) temperatures can be any temperature or temperatures inthe range of 45° C. to 95° C., e.g., 45, 50, 55, 60, 65, 70, 75, 80, 85,or 95° C. (or in between, e.g., 62° C., 68° C., 72° C., etc.) at one ormore (e.g., several) pHs in the range of 3 to 9, e.g., 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 (or in between) fora suitable period (time) of incubation, e.g., 1 minute, 5 minutes, 10minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, or60 minutes (or in between, e.g., 23 minutes, 37 minutes, etc.), suchthat the variant retains residual activity. However, longer periods ofincubation can also be used. The term “increased thermostability” can beused interchangeably with “improved thermostability”.

The increased thermostability of the variant relative to the parent canbe determined by differential scanning calorimetry (DSC) using methodsstandard in the art (see, for example, Sturtevant, 1987, Annual Reviewof Physical Chemistry 38: 463-488; Examples 9 and 17). The increasedthermostability of the variant relative to the parent can also bedetermined using protein thermal unfolding analysis (see, for example,Examples 10, 18, and 23 herein). The increased thermostability of thevariant relative to the parent can also be determined using any enzymeassay known in the art for GH61 polypeptides having cellulolyticenhancing activity to measure residual activity after a temperaturetreatment. See for example, WO 2005/074647, WO 2008/148131 WO2005/074656, WO 2010/065830, WO 2007/089290, WO 2009/085935, WO2009/085859, WO 2009/085864, WO 2009/085868, and WO 2008/151043, whichare incorporated herein by reference. Alternatively, the increasedthermostability of the variant relative to the parent can be determinedusing any application assay for the variant where the performance of thevariant is compared to the parent. For example, the application assaysdescribed in Examples 5, 12, and 13 can be used.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 20 to 326 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 19 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 18 to 239 of SEQID NO: 4 based on the SignalP program that predicts amino acids 1 to 17of SEQ ID NO: 4 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 20 to 258 of SEQ ID NO: 6 based on theSignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 6 are asignal peptide. In another aspect, the mature polypeptide is amino acids19 to 226 of SEQ ID NO: 8 based on the SignalP program that predictsamino acids 1 to 18 of SEQ ID NO: 8 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 20 to 304 of SEQ ID NO: 10based on the SignalP program that predicts amino acids 1 to 19 of SEQ IDNO: 10 are a signal peptide. In another aspect, the mature polypeptideis amino acids 16 to 317 of SEQ ID NO: 12 based on the SignalP programthat predicts amino acids 1 to 15 of SEQ ID NO: 12 are a signal peptide.In another aspect, the mature polypeptide is amino acids 22 to 249 ofSEQ ID NO: 14 based on the SignalP program that predicts amino acids 1to 21 of SEQ ID NO: 14 are a signal peptide. In another aspect, themature polypeptide is amino acids 20 to 249 of SEQ ID NO: 16 based onthe SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 16are a signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 232 of SEQ ID NO: 18 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 18 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 16 to 235 of SEQID NO: 20 based on the SignalP program that predicts amino acids 1 to 15of SEQ ID NO: 20 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 19 to 323 of SEQ ID NO: 22 based on theSignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 22 are asignal peptide. In another aspect, the mature polypeptide is amino acids16 to 310 of SEQ ID NO: 24 based on the SignalP program that predictsamino acids 1 to 15 of SEQ ID NO: 24 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 20 to 246 of SEQ ID NO: 26based on the SignalP program that predicts amino acids 1 to 19 of SEQ IDNO: 26 are a signal peptide. In another aspect, the mature polypeptideis amino acids 22 to 354 of SEQ ID NO: 28 based on the SignalP programthat predicts amino acids 1 to 21 of SEQ ID NO: 28 are a signal peptide.In another aspect, the mature polypeptide is amino acids 22 to 250 ofSEQ ID NO: 30 based on the SignalP program that predicts amino acids 1to 21 of SEQ ID NO: 30 are a signal peptide. In another aspect, themature polypeptide is amino acids 22 to 322 of SEQ ID NO: 32 based onthe SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 32are a signal peptide. In another aspect, the mature polypeptide is aminoacids 24 to 444 of SEQ ID NO: 34 based on the SignalP program thatpredicts amino acids 1 to 23 of SEQ ID NO: 34 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 26 to 253 of SEQID NO: 36 based on the SignalP program that predicts amino acids 1 to 25of SEQ ID NO: 36 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 18 to 246 of SEQ ID NO: 38 based on theSignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 38 are asignal peptide. In another aspect, the mature polypeptide is amino acids20 to 334 of SEQ ID NO: 40 based on the SignalP program that predictsamino acids 1 to 19 of SEQ ID NO: 40 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 18 to 227 of SEQ ID NO: 42based on the SignalP program that predicts amino acids 1 to 17 of SEQ IDNO: 42 are a signal peptide. In another aspect, the mature polypeptideis amino acids 20 to 223 of SEQ ID NO: 44 based on the SignalP programthat predicts amino acids 1 to 19 of SEQ ID NO: 44 are a signal peptide.In another aspect, the mature polypeptide is amino acids 22 to 368 ofSEQ ID NO: 46 based on the SignalP program that predicts amino acids 1to 21 of SEQ ID NO: 46 are a signal peptide. In another aspect, themature polypeptide is amino acids 25 to 330 of SEQ ID NO: 48 based onthe SignalP program that predicts amino acids 1 to 24 of SEQ ID NO: 48are a signal peptide. In another aspect, the mature polypeptide is aminoacids 17 to 236 of SEQ ID NO: 50 based on the SignalP program thatpredicts amino acids 1 to 16 of SEQ ID NO: 50 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 19 to 250 of SEQID NO: 52 based on the SignalP program that predicts amino acids 1 to 18of SEQ ID NO: 52 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 23 to 478 of SEQ ID NO: 54 based on theSignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 54 are asignal peptide. In another aspect, the mature polypeptide is amino acids17 to 230 of SEQ ID NO: 56 based on the SignalP program that predictsamino acids 1 to 16 of SEQ ID NO: 56 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 20 to 257 of SEQ ID NO: 58based on the SignalP program that predicts amino acids 1 to 19 of SEQ IDNO: 58 are a signal peptide. In another aspect, the mature polypeptideis amino acids 23 to 251 of SEQ ID NO: 60 based on the SignalP programthat predicts amino acids 1 to 22 of SEQ ID NO: 60 are a signal peptide.In another aspect, the mature polypeptide is amino acids 19 to 349 ofSEQ ID NO: 62 based on the SignalP program that predicts amino acids 1to 18 of SEQ ID NO: 62 are a signal peptide. In another aspect, themature polypeptide is amino acids 24 to 436 of SEQ ID NO: 64 based onthe SignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 64are a signal peptide. In another aspect, the mature polypeptide is aminoacids 21 to 344 of SEQ ID NO: 66 based on the SignalP program thatpredicts amino acids 1 to 23 of SEQ ID NO: 66 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 26 to 400 of SEQID NO: 68 based on the SignalP program that predicts amino acids 1 to 25of SEQ ID NO: 68 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 389 of SEQ ID NO: 70 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 70 are asignal peptide. In another aspect, the mature polypeptide is amino acids22 to 406 of SEQ ID NO: 72 based on the SignalP program that predictsamino acids 1 to 21 of SEQ ID NO: 72 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 20 to 427 of SEQ ID NO: 74based on the SignalP program that predicts amino acids 1 to 19 of SEQ IDNO: 74 are a signal peptide. In another aspect, the mature polypeptideis amino acids 18 to 267 of SEQ ID NO: 76 based on the SignalP programthat predicts amino acids 1 to 17 of SEQ ID NO: 76 are a signal peptide.In another aspect, the mature polypeptide is amino acids 21 to 273 ofSEQ ID NO: 78 based on the SignalP program that predicts amino acids 1to 20 of SEQ ID NO: 78 are a signal peptide. In another aspect, themature polypeptide is amino acids 23 to 272 of SEQ ID NO: 80 based onthe SignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 80are a signal peptide. In another aspect, the mature polypeptide is aminoacids 22 to 327 of SEQ ID NO: 82 based on the SignalP program thatpredicts amino acids 1 to 21 of SEQ ID NO: 82 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 23 to 274 of SEQID NO: 84 based on the SignalP program that predicts amino acids 1 to 22of SEQ ID NO: 84 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 322 of SEQ ID NO: 86 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 86 are asignal peptide. In another aspect, the mature polypeptide is amino acids18 to 234 of SEQ ID NO: 88 based on the SignalP program that predictsamino acids 1 to 17 of SEQ ID NO: 88 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 24 to 233 of SEQ ID NO: 90based on the SignalP program that predicts amino acids 1 to 23 of SEQ IDNO: 90 are a signal peptide. In another aspect, the mature polypeptideis amino acids 17 to 237 of SEQ ID NO: 92 based on the SignalP programthat predicts amino acids 1 to 16 of SEQ ID NO: 92 are a signal peptide.In another aspect, the mature polypeptide is amino acids 20 to 484 ofSEQ ID NO: 94 based on the SignalP program that predicts amino acids 1to 19 of SEQ ID NO: 94 are a signal peptide. In another aspect, themature polypeptide is amino acids 22 to 329 of SEQ ID NO: 96 based onthe SignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 96are a signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 227 of SEQ ID NO: 98 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 98 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 17 to 257 of SEQID NO: 100 based on the SignalP program that predicts amino acids 1 to16 of SEQ ID NO: 100 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 20 to 246 of SEQ ID NO: 102 based on theSignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 102 area signal peptide. In another aspect, the mature polypeptide is aminoacids 28 to 265 of SEQ ID NO: 104 based on the SignalP program thatpredicts amino acids 1 to 27 of SEQ ID NO: 104 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 16 to 310 of SEQID NO: 106 based on the SignalP program that predicts amino acids 1 to15 of SEQ ID NO: 106 are a signal peptide. In one aspect, the maturepolypeptide is amino acids 21 to 354 of SEQ ID NO: 108 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 108 area signal peptide. In another aspect, the mature polypeptide is aminoacids 22 to 267 of SEQ ID NO: 110 based on the SignalP program thatpredicts amino acids 1 to 21 of SEQ ID NO: 110 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 16 to 237 of SEQID NO: 112 based on the SignalP program that predicts amino acids 1 to15 of SEQ ID NO: 112 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 20 to 234 of SEQ ID NO: 114 based on theSignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 114 area signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 226 of SEQ ID NO: 116 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 116 are a signal peptide. Inone aspect, the mature polypeptide is amino acids 17 to 231 of SEQ IDNO: 118 based on the SignalP program that predicts amino acids 1 to 16of SEQ ID NO: 118 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 22 to 248 of SEQ ID NO: 120 based on theSignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 120 area signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 233 of SEQ ID NO: 122 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 122 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 21 to 243 of SEQID NO: 124 based on the SignalP program that predicts amino acids 1 to20 of SEQ ID NO: 124 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 363 of SEQ ID NO: 126 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 126 area signal peptide. In one aspect, the mature polypeptide is amino acids20 to 296 of SEQ ID NO: 128 based on the SignalP program that predictsamino acids 1 to 19 of SEQ ID NO: 128 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 16 to 318 of SEQ ID NO:130 based on the SignalP program that predicts amino acids 1 to 15 ofSEQ ID NO: 130 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 19 to 259 of SEQ ID NO: 132 based on theSignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 132 area signal peptide. In another aspect, the mature polypeptide is aminoacids 20 to 325 of SEQ ID NO: 134 based on the SignalP program thatpredicts amino acids 1 to 19 of SEQ ID NO: 134 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 19 to 298 of SEQID NO: 136 based on the SignalP program that predicts amino acids 1 to18 of SEQ ID NO: 136 are a signal peptide. In one aspect, the maturepolypeptide is amino acids 20 to 298 of SEQ ID NO: 138 based on theSignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 138 area signal peptide. In another aspect, the mature polypeptide is aminoacids 22 to 344 of SEQ ID NO: 140 based on the SignalP program thatpredicts amino acids 1 to 21 of SEQ ID NO: 140 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 20 to 330 of SEQID NO: 142 based on the SignalP program that predicts amino acids 1 to19 of SEQ ID NO: 142 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 19 to 216 of SEQ ID NO: 144 based on theSignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 144 area signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 490 of SEQ ID NO: 146 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 146 are a signal peptide. Inone aspect, the mature polypeptide is amino acids 21 to 306 of SEQ IDNO: 148 based on the SignalP program) that predicts amino acids 1 to 20of SEQ ID NO: 148 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 22 to 339 of SEQ ID NO: 150 based on theSignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 150 area signal peptide. In another aspect, the mature polypeptide is aminoacids 22 to 344 of SEQ ID NO: 152 based on the SignalP program thatpredicts amino acids 1 to 21 of SEQ ID NO: 152 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 23 to 408 of SEQID NO: 154 based on the SignalP program that predicts amino acids 1 to22 of SEQ ID NO: 154 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 19 to 234 of SEQ ID NO: 156 based on theSignalP program that predicts amino acids 1 to 18 of SEQ ID NO: 156 area signal peptide. In another aspect, the mature polypeptide is aminoacids 22 to 248 of SEQ ID NO: 158 based on the SignalP program thatpredicts amino acids 1 to 21 of SEQ ID NO: 158 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 21 to 242 of SEQID NO: 160 based on the SignalP program that predicts amino acids 1 to20 of SEQ ID NO: 160 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 23 to 334 of SEQ ID NO: 162 based on theSignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 162 area signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 230 of SEQ ID NO: 164 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 164 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 19 to 397 of SEQID NO: 166 based on the SignalP program that predicts amino acids 1 to18 of SEQ ID NO: 166 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 23 to 410 of SEQ ID NO: 168 based on theSignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 168 area signal peptide. In another aspect, the mature polypeptide is aminoacids 20 to 232 of SEQ ID NO: 170 based on the SignalP program thatpredicts amino acids 1 to 19 of SEQ ID NO: 170 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 21 to 266 of SEQID NO: 172 based on the SignalP program that predicts amino acids 1 to20 of SEQ ID NO: 172 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 24 to 324 of SEQ ID NO: 174 based on theSignalP program that predicts amino acids 1 to 23 of SEQ ID NO: 174 area signal peptide. In another aspect, the mature polypeptide is aminoacids 21 to 240 of SEQ ID NO: 176 based on the SignalP program thatpredicts amino acids 1 to 20 of SEQ ID NO: 176 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 21 to 225 of SEQID NO: 178 based on the SignalP program that predicts amino acids 1 to20 of SEQ ID NO: 178 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 16 to 235 of SEQ ID NO: 180 based on theSignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 180 area signal peptide. In another aspect, the mature polypeptide is aminoacids 20 to 336 of SEQ ID NO: 182 based on the SignalP program thatpredicts amino acids 1 to 19 of SEQ ID NO: 182 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 17 to 253 of SEQID NO: 184 based on the SignalP program that predicts amino acids 1 to16 of SEQ ID NO: 184 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 18 to 255 of SEQ ID NO: 186 based on theSignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 186 area signal peptide. In another aspect, the mature polypeptide is aminoacids 18 to 225 of SEQ ID NO: 188 based on the SignalP program thatpredicts amino acids 1 to 17 of SEQ ID NO: 188 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 16 to 237 of SEQID NO: 190 based on the SignalP program that predicts amino acids 1 to15 of SEQ ID NO: 190 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 18 to 227 of SEQ ID NO: 192 based on theSignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 192 area signal peptide. In another aspect, the mature polypeptide is aminoacids 22 to 315 of SEQ ID NO: 194 based on the SignalP program thatpredicts amino acids 1 to 21 of SEQ ID NO: 194 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 21 to 439 of SEQID NO: 196 based on the SignalP program that predicts amino acids 1 to20 of SEQ ID NO: 196 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 18 to 246 of SEQ ID NO: 198 based on theSignalP program that predicts amino acids 1 to 17 of SEQ ID NO: 198 area signal peptide. In another aspect, the mature polypeptide is aminoacids 19 to 324 of SEQ ID NO: 200 based on the SignalP program thatpredicts amino acids 1 to 18 of SEQ ID NO: 200 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 21 to 242 of SEQID NO: 202 based on the SignalP program that predicts amino acids 1 to20 of SEQ ID NO: 202 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 16 to 306 of SEQ ID NO: 204 based on theSignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 204 area signal peptide. In another aspect, the mature polypeptide is aminoacids 20 to 252 of SEQ ID NO: 206 based on the SignalP program thatpredicts amino acids 1 to 19 of SEQ ID NO: 206 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 20 to 344 of SEQID NO: 208 based on the SignalP program that predicts amino acids 1 to19 of SEQ ID NO: 208 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 22 to 347 of SEQ ID NO: 210 based on theSignalP program that predicts amino acids 1 to 21 of SEQ ID NO: 210 area signal peptide. In another aspect, the mature polypeptide is aminoacids 23 to 334 of SEQ ID NO: 212 based on the SignalP program thatpredicts amino acids 1 to 22 of SEQ ID NO: 212 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 24 to 366 of SEQID NO: 214 based on the SignalP program that predicts amino acids 1 to23 of SEQ ID NO: 214 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 21 to 364 of SEQ ID NO: 216 based on theSignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 216 area signal peptide. It is known in the art that a host cell may produce amixture of two of more different mature polypeptides (i.e., with adifferent C-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO: 1based on the SignalP program (Nielsen et al., 1997, supra) that predictsnucleotides 330 to 387 of SEQ ID NO: 1 encode a signal peptide. Inanother aspect, the mature polypeptide coding sequence is nucleotides 98to 821 of SEQ ID NO: 3 based on the SignalP program that predictsnucleotides 47 to 97 of SEQ ID NO: 3 encode a signal peptide. In anotheraspect, the mature polypeptide coding sequence is nucleotides 126 to 978of SEQ ID NO: 5 based on the SignalP program that predicts nucleotides69 to 125 of SEQ ID NO: 5 encode a signal peptide. In another aspect,the mature polypeptide coding sequence is nucleotides 55 to 678 of SEQID NO: 7 based on the SignalP program that predicts nucleotides 1 to 54of SEQ ID NO: 7 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is nucleotides 58 to 912 of SEQ ID NO: 9based on the SignalP program that predicts nucleotides 1 to 57 of SEQ IDNO: 9 encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 951 of SEQ ID NO: 11 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 11encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 796 of SEQ ID NO: 13 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 13encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 77 to 766 of SEQ ID NO: 15 based on theSignalP program that predicts nucleotides 20 to 76 of SEQ ID NO: 15encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 921 of SEQ ID NO: 17 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 17encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 851 of SEQ ID NO: 19 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 19encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1239 of SEQ ID NO: 21 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 21encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 1250 of SEQ ID NO: 23 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 23encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 811 of SEQ ID NO: 25 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 25encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1112 of SEQ ID NO: 27 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 27encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 859 of SEQ ID NO: 29 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 29encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1018 of SEQ ID NO: 31 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 31encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 70 to 1483 of SEQ ID NO: 33 based on theSignalP program that predicts nucleotides 1 to 69 of SEQ ID NO: 33encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 76 to 832 of SEQ ID NO: 35 based on theSignalP program that predicts nucleotides 1 to 75 of SEQ ID NO: 35encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 875 of SEQ ID NO: 37 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 37encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1250 of SEQ ID NO: 39 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 39encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 795 of SEQ ID NO: 41 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 41encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 974 of SEQ ID NO: 43 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 43encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1104 of SEQ ID NO: 45 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 45encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 73 to 990 of SEQ ID NO: 47 based on theSignalP program that predicts nucleotides 1 to 72 of SEQ ID NO: 47encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 49 to 1218 of SEQ ID NO: 49 based on theSignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 49encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 930 of SEQ ID NO: 51 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 51encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 1581 of SEQ ID NO: 53 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 53encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 49 to 865 of SEQ ID NO: 55 based on theSignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 55encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1065 of SEQ ID NO: 57 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 57encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 868 of SEQ ID NO: 59 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 59encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1099 of SEQ ID NO: 61 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 61encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 70 to 1490 of SEQ ID NO: 63 based on theSignalP program that predicts nucleotides 1 to 69 of SEQ ID NO: 63encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 1032 of SEQ ID NO: 65 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 65encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 76 to 1200 of SEQ ID NO: 67 based on theSignalP program that predicts nucleotides 1 to 75 of SEQ ID NO: 67encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 1167 of SEQ ID NO: 69 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 69encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1218 of SEQ ID NO: 71 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 71encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1281 of SEQ ID NO: 73 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 73encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 801 of SEQ ID NO: 75 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 75encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 819 of SEQ ID NO: 77 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 77encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 869 of SEQ ID NO: 79 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 79encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1036 of SEQ ID NO: 81 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 81encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 878 of SEQ ID NO: 83 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 83encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 966 of SEQ ID NO: 85 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 85encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 702 of SEQ ID NO: 87 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 87encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 70 to 699 of SEQ ID NO: 89 based on theSignalP program that predicts nucleotides 1 to 69 of SEQ ID NO: 89encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 49 to 711 of SEQ ID NO: 91 based on theSignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 91encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1452 of SEQ ID NO: 93 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 93encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1018 of SEQ ID NO: 95 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 95encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 818 of SEQ ID NO: 97 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 97encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 49 to 1117 of SEQ ID NO: 99 based on theSignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 99encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 875 of SEQ ID NO: 101 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 101encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 82 to 1064 of SEQ ID NO: 103 based on theSignalP program that predicts nucleotides 1 to 81 of SEQ ID NO: 103encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 1032 of SEQ ID NO: 105 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 105encode a signal peptide. In one aspect, the mature polypeptide codingsequence is nucleotides 61 to 1062 of SEQ ID NO: 107 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 107encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 801 of SEQ ID NO: 109 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 109encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 840 of SEQ ID NO: 111 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 111encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 702 of SEQ ID NO: 113 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 113encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 750 of SEQ ID NO: 115 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 115encode a signal peptide. In one aspect, the mature polypeptide codingsequence is nucleotides 49 to 851 of SEQ ID NO: 117 based on the SignalPprogram that predicts nucleotides 1 to 48 of SEQ ID NO: 117 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 64 to 860 of SEQ ID NO: 119 based on the SignalPprogram that predicts nucleotides 1 to 63 of SEQ ID NO: 119 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 52 to 830 of SEQ ID NO: 121 based on the SignalPprogram that predicts nucleotides 1 to 51 of SEQ ID NO: 121 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 61 to 925 of SEQ ID NO: 123 based on the SignalPprogram that predicts nucleotides 1 to 60 of SEQ ID NO: 123 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 61 to 1089 of SEQ ID NO: 125 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 125encode a signal peptide. In one aspect, the mature polypeptide codingsequence is nucleotides 58 to 1083 of SEQ ID NO: 127 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 127encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 1029 of SEQ ID NO: 129 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 129encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1110 of SEQ ID NO: 131 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 131encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1100 of SEQ ID NO: 133 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 133encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1036 of SEQ ID NO: 135 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 135encode a signal peptide. In one aspect, the mature polypeptide codingsequence is nucleotides 58 to 1022 of SEQ ID NO: 137 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 137encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1032 of SEQ ID NO: 139 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 139encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1054 of SEQ ID NO: 141 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 141encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 769 of SEQ ID NO: 143 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 143encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 1533 of SEQ ID NO: 145 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 145encode a signal peptide. In one aspect, the mature polypeptide codingsequence is nucleotides 61 to 918 of SEQ ID NO: 147 based on the SignalPprogram that predicts nucleotides 1 to 60 of SEQ ID NO: 147 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 64 to 1089 of SEQ ID NO: 149 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 149encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1086 of SEQ ID NO: 151 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 151encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 1395 of SEQ ID NO: 153 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 155encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 899 of SEQ ID NO: 155 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 155encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 807 of SEQ ID NO: 157 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 157encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 726 of SEQ ID NO: 159 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 159encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 1078 of SEQ ID NO: 161 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 161encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 872 of SEQ ID NO: 163 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 163encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1191 of SEQ ID NO: 165 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 165encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 1230 of SEQ ID NO: 167 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 167encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 696 of SEQ ID NO: 169 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 169encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 798 of SEQ ID NO: 171 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 171encode a signal peptide. In one aspect, the mature polypeptide codingsequence is nucleotides 70 to 972 of SEQ ID NO: 173 based on the SignalPprogram that predicts nucleotides 1 to 69 of SEQ ID NO: 173 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 61 to 1112 of SEQ ID NO: 175 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 175encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 985 of SEQ ID NO: 177 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 177encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 856 of SEQ ID NO: 179 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 179encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1008 of SEQ ID NO: 181 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 181encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 49 to 1312 of SEQ ID NO: 183 based on theSignalP program that predicts nucleotides 1 to 48 of SEQ ID NO: 183encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 921 of SEQ ID NO: 185 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 185encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 739 of SEQ ID NO: 187 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 187encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 898 of SEQ ID NO: 189 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 189encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 941 of SEQ ID NO: 191 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 191encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 945 of SEQ ID NO: 193 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 193encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 1377 of SEQ ID NO: 195 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 195encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 818 of SEQ ID NO: 197 based on theSignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 197encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1122 of SEQ ID NO: 199 based on theSignalP program that predicts nucleotides 1 to 54 of SEQ ID NO: 199encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 60 to 1034 of SEQ ID NO: 201 based on theSignalP program that predicts nucleotides 1 to 61 of SEQ ID NO: 201encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 46 to 1197 of SEQ ID NO: 203 based on theSignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 203encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 756 of SEQ ID NO: 205 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 205encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1032 of SEQ ID NO: 207 based on theSignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 207encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 64 to 1041 of SEQ ID NO: 209 based on theSignalP program that predicts nucleotides 1 to 63 of SEQ ID NO: 209encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 67 to 1002 of SEQ ID NO: 211 based on theSignalP program that predicts nucleotides 1 to 66 of SEQ ID NO: 211encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 70 to 1098 of SEQ ID NO: 213 based on theSignalP program that predicts nucleotides 1 to 69 of SEQ ID NO: 213encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 61 to 1088 of SEQ ID NO: 215 based on theSignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 215encode a signal peptide. In each of the aspects above, the term “maturepolypeptide coding sequence” shall be understood to include the cDNAsequence of the genomic DNA sequence or the genomic DNA sequence of thecDNA sequence.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent GH61 polypeptide: The term “parent” or “parent GH61polypeptide” means a GH61 polypeptide to which an alteration is made toproduce the GH61 polypeptide variants of the present invention. Theparent may be a naturally occurring (wild-type) polypeptide or a variantor fragment thereof.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide or variant thereof that catalyzes the enhancement of thehydrolysis of a cellulosic material by enzyme having cellulolyticactivity. For purposes of the present invention, cellulolytic enhancingactivity is determined by measuring the increase in reducing sugars orthe increase of the total of cellobiose and glucose from the hydrolysisof a cellulosic material by cellulolytic enzyme under the followingconditions: 1-50 mg of total protein/g of cellulose in pretreated cornstover (PCS), wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of a GH61polypeptide or variant thereof for 1-7 days at a suitable temperaturesuch as 40° C.-80° C., e.g., 50° C., 55° C., 60° C., 65° C., or 70° C.,and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0,compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity. Another assay for determining the cellulolyticenhancing activity of a GH61 polypeptide or variant thereof is toincubate the GH61 polypeptide or variant with 0.5% phosphoric acidswollen cellulose (PASC), 100 mM sodium acetate pH 5, 1 mM MnSO₄, 0.1%gallic acid, 0.025 mg/ml of Aspergillus fumigatus beta-glucosidase, and0.01% TRITON® X100 for 24-96 hours at 40° C. followed by determinationof the glucose released from the PASC.

The GH61 polypeptides or variants thereof having cellulolytic enhancingactivity enhance the hydrolysis of a cellulosic material catalyzed byenzyme having cellulolytic activity by reducing the amount ofcellulolytic enzyme required to reach the same degree of hydrolysispreferably at least 1.01-fold, e.g., at least 1.05-fold, at least1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 20-fold.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows: (IdenticalResidues×100)/(Length of Alignment−Total Number of Gaps in Alignment)For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows: (IdenticalDeoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence, wherein the subsequence encodes afragment having cellulolytic enhancing activity. In one aspect, asubsequence contains at least 85% of the nucleotides, e.g., at least 90%of the nucleotides or at least 95% of the nucleotides of the maturepolypeptide coding sequence of a GH61 polypeptide.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition. The variants of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the cellulolytic enhancingactivity of their parent GH61 polypeptides.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type GH61 polypeptide: The term “wild-type” GH61 polypeptide meansa GH61 polypeptide expressed by a naturally occurring microorganism,such as a bacterium, yeast, or filamentous fungus found in nature.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Recent progress in the assays of xylanolytic enzymes,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 30 is used to determine the corresponding amino acidresidue in another GH61 polypeptide. The amino acid sequence of anotherGH61 polypeptide is aligned with the mature polypeptide disclosed in SEQID NO: 30, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 30 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.Numbering of the amino acid positions is based on the full-lengthpolypeptide (e.g., including the signal peptide) of SEQ ID NO: 30wherein position 1 is the first amino acid of the signal peptide (e.g.,Met).

Identification of the corresponding amino acid residue in another GH61polypeptide can be determined by alignment of multiple polypeptidesequences using several computer programs including, but not limited toMUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797); MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

For example, the position corresponding to position 105 of theAspergillus fumigatus GH61 polypeptide (SEQ ID NO: 30) is position 109in the Penicillium emersonii GH61 polypeptide (SEQ ID NO: 36), position105 in the Thermoascus aurantiacus GH61 polypeptide (SEQ ID NO: 14), andposition 103 in the Aspergillus aculeatus GH61 polypeptide (SEQ ID NO:68); the position corresponding to position 188 of the Aspergillusfumigatus GH61 polypeptide is position 192 in the Penicillium emersoniiGH61 polypeptide, position 188 in the Thermoascus aurantiacus GH61polypeptide, and position 186 in the Aspergillus aculeatus GH61polypeptide; the position corresponding to position 154 of theAspergillus fumigatus GH61 polypeptide is position 152 in theAspergillus aculeatus GH61 polypeptide; and the position correspondingto position 189 of the Aspergillus fumigatus GH61 polypeptide isposition 193 in the Penicillium emersonii GH61 polypeptide and position187 in the Aspergillus aculeatus GH61 polypeptide.

When another GH61 polypeptide has diverged from the mature polypeptideof SEQ ID NO: 30 such that traditional sequence-based comparison failsto detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol.295: 613-615), other pairwise sequence comparison algorithms can beused. Greater sensitivity in sequence-based searching can be attainedusing search programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the GH61 polypeptide variants of the present invention,the nomenclature described below is adapted for ease of reference. Theaccepted IUPAC single letter or three letter amino acid abbreviation isemployed.

Substitutions.

For an amino acid substitution, the following nomenclature is used:Original amino acid, position, substituted amino acid. Accordingly, thesubstitution of threonine at position 226 with alanine is designated as“Thr226Ala” or “T226A”. Multiple mutations are separated by additionmarks (“+”), e.g., “Gly205Arg+Ser411Phe” or “G205R+S411F”, representingsubstitutions at positions 205 and 411 of glycine (G) with arginine (R)and serine (S) with phenylalanine (F), respectively.

Deletions.

For an amino acid deletion, the following nomenclature is used: Originalamino acid, position, *. Accordingly, the deletion of glycine atposition 195 is designated as “Gly195*” or “G195*”. Multiple deletionsare separated by addition marks (“+”), e.g., “Gly195*+Ser411*” or“G195*+S411*”.

Insertions.

For an amino acid insertion, the following nomenclature is used:Original amino acid, position, original amino acid, inserted amino acid.Accordingly the insertion of lysine after glycine at position 195 isdesignated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple Substitutions.

Variants comprising multiple substitutions are separated by additionmarks (“+”), e.g., “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing asubstitution of arginine and glycine at positions 170 and 195 withtyrosine and glutamic acid, respectively.

Different Substitutions.

Where different substitutions can be introduced at a position, thedifferent substitutions are separated by a comma, e.g., “Arg170Tyr,Glu”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”,“Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated GH61 polypeptide variants,comprising a substitution at one or more (e.g., several) positionscorresponding to positions 105, 154, 188, 189, 216, and 229 of themature polypeptide of SEQ ID NO: 30, wherein the variants havecellulolytic enhancing activity.

Variants

In an embodiment, the variant has a sequence identity of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%, tothe amino acid sequence of the parent GH61 polypeptide.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216.

In one aspect, the number of substitutions in the variants of thepresent invention is 1-6, e.g., 1, 2, 3, 4, 5, or 6 substitutions.

In another aspect, a variant comprises a substitution at one or more(e.g., several) positions corresponding to positions 105, 154, 188, 189,216, and 229. In another aspect, a variant comprises a substitution attwo positions corresponding to any of positions 105, 154, 188, 189, 216,and 229. In another aspect, a variant comprises a substitution at threepositions corresponding to any of positions 105, 154, 188, 189, 216, and229. In another aspect, a variant comprises a substitution at fourpositions corresponding to any of positions 105, 154, 188, 189, 216, and229. In another aspect, a variant comprises a substitution at fivepositions corresponding to any of positions 105, 154, 188, 189, 216, and229. In another aspect, a variant comprises a substitution at eachposition corresponding to positions 105, 154, 188, 189, 216, and 229.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 105. In another aspect, theamino acid at a position corresponding to position 105 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Pro or Lys. Inanother aspect, the variant comprises or consists of the substitutionE105P or E105K of the mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 154. In another aspect, theamino acid at a position corresponding to position 154 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile or Leu. Inanother aspect, the variant comprises or consists of the substitutionE154I or E154L of the mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 188. In another aspect, theamino acid at a position corresponding to position 188 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Met, Phe, orTrp. In another aspect, the variant comprises or consists of thesubstitution G188A, G188F, G188M, or G188W of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 189. In another aspect, theamino acid at a position corresponding to position 189 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with His or Lys. Inanother aspect, the variant comprises or consists of the substitutionN189H or N189K of the mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 216. In another aspect, theamino acid at a position corresponding to position 216 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu or Tyr. Inanother aspect, the variant comprises or consists of the substitutionA216L or A216Y of the mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 229. In another aspect, theamino acid at a position corresponding to position 229 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Trp, His, Ile, orTyr. In another aspect, the variant comprises or consists of thesubstitution K229W, K229H, K229I, or K229Y of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105 and 154, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105 and 188, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105 and 189, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105 and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105 and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154 and 188, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154 and 189, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154 and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154 and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188 and 189, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188 and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188 and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 189 and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 189 and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 216 and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, and 188, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, and 189, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 188, and 189, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 188, and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 188, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 189, and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 189, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 216, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, and 189, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 189, and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 189, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 216, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188, 189, and 216, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188, 189, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188, 216, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 189, 216, and 229, such as thosedescribed above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, and 189, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, and 216, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, and 229, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 189, and 216, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 189, and 229, such asthose described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 105,154, 216, and 229, such as those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 188, 189, and 216, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 188, 189, and 229, such asthose described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 105,188, 216, and 229, such as those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 189, 216, and 229, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, 189, and 216, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, 189, and 229, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, 216, and 229, such asthose described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 154,189, 216, and 229, such as those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 188, 189, 216, and 229, such asthose described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, 189, and 216, suchas those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, 189, and 229, suchas those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, 216, and 229, suchas those described above. In another aspect, the variant comprises orconsists of substitutions at positions corresponding to positions 105,154, 189, 216, and 229, such as those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 188, 189, 216, and 229, suchas those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 154, 188, 189, 216, and 229, suchas those described above.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 105, 154, 188, 189, 216, and 229,such as those described above.

In another aspect, the variant comprises or consists of one or more(e.g., several) substitutions selected from the group consisting ofE105P,K; E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y, or the one ormore (e.g., several) substitutions selected from the group consisting ofE105P,K; E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y at positionscorresponding to SEQ ID NO: 30 in other GH61 polypeptides describedherein.

In each of the aspects below, the variant comprises or consists of theone or more (e.g., several) substitutions described below at positionscorresponding to SEQ ID NO: 30 in other GH61 polypeptides describedherein.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K and E154I,L of the mature polypeptide of SEQ IDNO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K and G188A,F,M,W of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K and N189H,K of the mature polypeptide of SEQ IDNO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K and A216L,Y of the mature polypeptide of SEQ IDNO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K and K229W,H,I,Y of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L and G188A,F,M,W of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L and N189H,K of the mature polypeptide of SEQ IDNO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L and A216L,Y of the mature polypeptide of SEQ IDNO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L and K229W,H,I,Y of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W and N189H,K of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W and A216L,Y of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W and K229W,H,I,Y of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions N189H,K and A216L,Y of the mature polypeptide of SEQ IDNO: 30.

In another aspect, the variant comprises or consists of thesubstitutions N189H,K and K229W,H,I,Y of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions A216L,Y and K229W,H,I,Y of the mature polypeptide of SEQID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; and G188A,F,M,W of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; and N189H,K of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; and A216L,Y of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; and N189H,K of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; N189H,K; and A216L,Y of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; N189H,K; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; and N189H,K of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; N189H,K; and A216L,Y of the mature polypeptide ofSEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; N189H,K; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W; N189H,K; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W; N189H,K; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions N189H,K; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; and N189H,K of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; N189H,K; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; N189H,K; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; N189H,K; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; N189H,K; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; A216L,Y; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; N189H,K; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; N189H,K; and A216L,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; N189H,K; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and A216L,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and K229W,H,I,Y ofthe mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y ofthe mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y of themature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y ofthe mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E154I,L; G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y ofthe mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; A216L,Y; andK229W,H,I,Y of the mature polypeptide of SEQ ID NO: 30.

In another aspect, the variant comprises or consists of thesubstitutions D105K,P of the mature polypeptide of SEQ ID NO: 14. Inanother aspect, the variant comprises or consists of the substitutionsQ188W,F,M of the mature polypeptide of SEQ ID NO: 14.

In another aspect, the variant comprises or consists of the substitutionD109P,K of the mature polypeptide of SEQ ID NO: 36. In another aspect,the variant comprises or consists of the substitution N192A,W,M of themature polypeptide of SEQ ID NO: 36. In another aspect the variantcomprises or consists of the substitution N193K,H of the maturepolypeptide of SEQ ID NO: 36. In another aspect, the variant comprisesor consists of the substitution D109P,K of the mature polypeptide of SEQID NO: 36. In another aspect, the substitution is N192A,W,M of themature polypeptide of SEQ ID NO: 36. In another aspect, the variantcomprises or consists of the substitution N193K,H of the maturepolypeptide of SEQ ID NO: 36.

In another aspect, the variant comprises or consists of the substitutionD103K,P of the mature polypeptide of SEQ ID NO: 68. In another aspect,the variant comprises or consists of the substitution N1521,L of themature polypeptide of SEQ ID NO: 68. In another aspect the variantcomprises or consists of the substitution G186A,F,M,W of the maturepolypeptide of SEQ ID NO: 68. In another aspect, the variant comprisesor consists of the substitution N187H,K of the mature polypeptide of SEQID NO: 68.

The variants may further comprise one or more additional alterations,e.g., substitutions, insertions, or deletions at one or more (e.g.,several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

The variants of the present invention may further comprise asubstitution at one or more (e.g., several) positions corresponding topositions 111, 152, 155, and 162 of the mature polypeptide of SEQ ID NO:30, wherein the variants have cellulolytic enhancing activity (WO2012/044835).

In one aspect, the number of additional substitutions in the variants ofthe present invention is 1-4, such as 1, 2, 3, or 4 substitutions.

In another aspect, the variant further comprises a substitution at oneor more (e.g., several) positions corresponding to positions 111, 152,155, and 162. In another aspect, the variant further comprises asubstitution at two positions corresponding to any of positions 111,152, 155, and 162. In another aspect, the variant further comprises asubstitution at three positions corresponding to any of positions 111,152, 155, and 162. In another aspect, the variant further comprises asubstitution at each position corresponding to positions 111, 152, 155,and 162.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 111. In another aspect, the aminoacid at a position corresponding to position 111 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect,the variant further comprises the substitution L111V of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 152. In another aspect, the aminoacid at a position corresponding to position 152 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ser. In another aspect,the variant further comprises the substitution D152S of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 155. In another aspect, the aminoacid at a position corresponding to position 155 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect,the variant further comprises the substitution M155L of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 162. In another aspect, the aminoacid at a position corresponding to position 162 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Trp. In another aspect,the variant further comprises the substitution A162W of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111 and 152, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111 and 155, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111 and 162, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 152 and 155, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 152 and 162, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 155 and 162, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111, 152, and 155, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111, 152, and 162, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111, 155, and 162, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 152, 155, and 162, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 111, 152, 155, and 162, such asthose described above. In another aspect, the variant further comprisesone or more (e.g., several) substitutions selected from the groupconsisting of L111V, D152S, M155L, and A162W, or the one or more (e.g.,several) substitutions selected from the group consisting of L111V,D152S, M155L, and A162W at positions corresponding to SEQ ID NO: 30 inother GH61 polypeptides described herein.

In another aspect, the variant comprises substitutions L111V, D152S,M155L, A162W, and G188A, or the same substitutions at correspondingpositions thereof. In another aspect, the variant comprisessubstitutions L111V, D152S, M155L, A162W, G188F, and K229W, or the samesubstitutions at corresponding positions thereof. In another aspect, thevariant comprises substitutions L111V, D152S, M155L, A162W, and K229W,or corresponding substitutions thereof. In another aspect, the variantcomprises substitutions L111V, D152S, M155L, A162W, A216Y, and K229W, orthe same substitutions at corresponding positions thereof. In anotheraspect, the variant comprises substitutions L111V, D152S, M155L, A162W,N189K, and K229W, or the same substitutions at corresponding positionsthereof. In another aspect, the variant comprises substitutions L111V,D152S, M155L, A162W, and N189K, or the same substitutions atcorresponding positions thereof. In another aspect, the variantcomprises substitutions L111V, D152S, M155L, A162W, and G188W, or thesame substitutions at corresponding positions thereof.

In each of the aspects below, the variant further comprises the one ormore (e.g., several) substitutions described below at positionscorresponding to SEQ ID NO: 30 in other GH61 polypeptides describedherein.

In another aspect, the variant further comprises the substitutionsL111V+D152S of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsL111V+M155L of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsL111V+A162W of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsD152S+M155L of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsD152S+A162W of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsM155L+A162W of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsL111V+D152S+M155L of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsL111V+D152S+A162W of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsL111V+M155L+A162W of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsD152S+M155L+A162W of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsL111V+D152S+M155L+A162W of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In each of the aspects above, the variants of the present invention mayfurther comprise a substitution at one or more (e.g., several) positionscorresponding to positions 96, 98, 200, 202, and 204 of the maturepolypeptide of SEQ ID NO: 30, wherein the variants have cellulolyticenhancing activity (WO 2012/044836).

In one aspect, the number of additional substitutions in the variants ofthe present invention is 1-5, such as 1, 2, 3, 4, or 5 substitutions.

In another aspect, the variant further comprises a substitution at oneor more (e.g., several) positions corresponding to positions 96, 98,200, 202, and 204. In another aspect, the variant further comprises asubstitution at two positions corresponding to any of positions 96, 98,200, 202, and 204. In another aspect, the variant further comprises asubstitution at three positions corresponding to any of positions 96,98, 200, 202, and 204. In another aspect, the variant further comprisesa substitution at four positions corresponding to any of positions 96,98, 200, 202, and 204. In another aspect, the variant further comprisesa substitution at each position corresponding to positions 96, 98, 200,202, and 204.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 96. In another aspect, the amino acidat a position corresponding to position 96 is substituted with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Val. In another aspect, thevariant further comprises the substitution 196V of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 98. In another aspect, the amino acidat a position corresponding to position 98 is substituted with Ala, Arg,Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect, thevariant further comprises the substitution F98L of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 200. In another aspect, the aminoacid at a position corresponding to position 200 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ile. In another aspect,the variant further comprises the substitution F200I of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 202. In another aspect, the aminoacid at a position corresponding to position 202 is substituted withAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Leu. In another aspect,the variant further comprises the substitution I202L of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises a substitution at aposition corresponding to position 204. In another aspect, the aminoacid at a position corresponding to position 204 is substituted withAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Val. In another aspect,the variant further comprises the substitution I204V of the maturepolypeptide of SEQ ID NO: 30.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96 and 98, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96 and 200, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96 and 202, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96 and 204, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98 and 200, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98 and 202, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98 and 204, such as those describedabove.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 200 and 202, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 200 and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 202 and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 98, and 200, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 98, and 202, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 98, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 200, and 202, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 200, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 202, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98, 200, and 202, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98, 200, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 200, 202, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98, 202, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 98, 200, and 202, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 200, 202, and 204, such asthose described above. In another aspect, the variant further comprisessubstitutions at positions corresponding to positions 96, 98, 202, and204, such as those described above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 98, 200, and 204, such as thosedescribed above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 98, 200, 202, and 204, such asthose described above.

In another aspect, the variant further comprises substitutions atpositions corresponding to positions 96, 98, 200, 202, and 204, such asthose described above.

In another aspect, the variant further comprises one or more (e.g.,several) substitutions selected from the group consisting of I196V,F98L, F200I, I202L, and I204V, or the one or more (e.g., several)substitutions selected from the group consisting of I196V, F98L, F200I,I202L, and I204V at positions corresponding to SEQ ID NO: 30 in otherGH61 polypeptides described herein.

In each of the aspects below, the variant further comprises the one ormore (e.g., several) substitutions described below at positionscorresponding to SEQ ID NO: 30 in other GH61 polypeptides describedherein.

In another aspect, the variant further comprises the substitutionsI96V+F98L of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F200I of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+I202L of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+I204V of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+F200I of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+I202L of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+I204V of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsF200I+I202L of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsF200I+I204V of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsI202L+I204V of the mature polypeptide of SEQ ID NO: 30, or correspondingsubstitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+F200I of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+I202L of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F200I+I202L of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F200I+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+I202L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+F200I+I202L of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+F200I+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsF200I+I202L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+I202L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+F200I+I202L of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F200I+I202L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+I202L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+F200I+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsF98L+F200I+I202L+I204V of the mature polypeptide of SEQ ID NO: 30, orcorresponding substitutions thereof.

In another aspect, the variant further comprises the substitutionsI96V+F98L+F200I+I202L+I204V of the mature polypeptide of SEQ ID NO: 30,or corresponding substitutions thereof.

The variants may consist of at least 85% of the amino acid residues,e.g., at least 90% of the amino acid residues or at least 95% of theamino acid residues of the mature polypeptides of the correspondingparent GH61 polypeptides.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett.

309: 59-64. The identity of essential amino acids can also be inferredfrom an alignment with a related polypeptide. Essential amino acids inGH61 polypeptides correspond to positions 22, 107, 194, and/or 196 ofthe mature polypeptide of SEQ ID NO: 30.

In an embodiment, the variants have increased thermostability comparedto their parent GH61 polypeptides.

In one aspect, the thermostability of the variant relative to the parentis determined at pH 3.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 3.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 3.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.0 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.5 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.5 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.5 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.5 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 3.5 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.5 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.5 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.5 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.5 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 3.5 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 3.5 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 3.5 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 3.5 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 3.5 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 4.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 4.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.0 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.5 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.5 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.5 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.5 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 4.5 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.5 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.5 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.5 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.5 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 4.5 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 4.5 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 4.5 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 4.5 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 4.5 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 5.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 5.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.0 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.5 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.5 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.5 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.5 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 5.5 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.5 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.5 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.5 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.5 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 5.5 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 5.5 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 5.5 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 5.5 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 5.5 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 6.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 6.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.0 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.5 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.5 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.5 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.5 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 6.5 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.5 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.5 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.5 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.5 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 6.5 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 6.5 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 6.5 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 6.5 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 6.5 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 7.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 7.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.0 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.5 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.5 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.5 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.5 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 7.5 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.5 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.5 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.5 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.5 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 7.5 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 7.5 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 7.5 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 7.5 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 7.5 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 8.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 8.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.0 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.5 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.5 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.5 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.5 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 8.5 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.5 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.5 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.5 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.5 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 8.5 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 8.5 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 8.5 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 8.5 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 8.5 and 95° C.

In another aspect, the thermostability of the variant relative to theparent is determined at pH 9.0 and 45° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 9.0 and 50° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 9.0 and 55° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 9.0 and 60° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 9.0 and 62° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 9.0 and 65° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 9.0 and 68° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 9.0 and 70° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 9.0 and 72° C. In another aspect, the thermostabilityof the variant relative to the parent is determined at pH 9.0 and 75° C.In another aspect, the thermostability of the variant relative to theparent is determined at pH 9.0 and 80° C. In another aspect, thethermostability of the variant relative to the parent is determined atpH 9.0 and 85° C. In another aspect, the thermostability of the variantrelative to the parent is determined at pH 9.0 and 90° C. In anotheraspect, the thermostability of the variant relative to the parent isdetermined at pH 9.0 and 95° C.

In each of the aspects above, the thermostability of the variantrelative to the parent can be determined by incubating the variant andparent for 1 minute. In each of the aspects above, the thermostabilityof the variant relative to the parent can be determined by incubatingthe variant and parent for 5 minutes. In each of the aspects above, thethermostability of the variant relative to the parent can be determinedby incubating the variant and parent for 10 minutes. In each of theaspects above, the thermostability of the variant relative to the parentcan be determined by incubating the variant and parent for 15 minutes.In each of the aspects above, the thermostability of the variantrelative to the parent can be determined by incubating the variant andparent for 20 minutes. In each of the aspects above, the thermostabilityof the variant relative to the parent can be determined by incubatingthe variant and parent for 25 minutes. In each of the aspects above, thethermostability of the variant relative to the parent can be determinedby incubating the variant and parent for 30 minutes. In each of theaspects above, the thermostability of the variant relative to the parentcan be determined by incubating the variant and parent for 45 minutes.In each of the aspects above, the thermostability of the variantrelative to the parent can be determined by incubating the variant andparent for 60 minutes. A time period longer than 60 minutes can also beused.

In one aspect, the thermostability of the variant having cellulolyticenhancing activity is increased at least 1.01-fold, e.g., at least1.05-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, atleast 1.4-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold,at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold,at least 25-fold, at least 50-fold, at least 75-fold, or at least100-fold compared to the parent.

Parent GH61 Polypeptides

The parent GH61 polypeptide may be any GH61 polypeptide havingcellulolytic enhancing activity.

The parent GH61 polypeptide may be (a) a polypeptide having at least 60%sequence identity to the mature polypeptide of SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195,197, 199, 201, 203, 205, 207, 209, 211, 213, or 215; (b) a polypeptideencoded by a polynucleotide that hybridizes under at least lowstringency conditions with (i) the mature polypeptide coding sequence of1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215, or (ii)the full-length complement of (i); or (c) a polypeptide encoded by apolynucleotide having at least 60% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, or 215.

In one aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216 of at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have cellulolytic enhancing activity.

In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 from themature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, or 216.

In another aspect, the parent comprises or consists of the maturepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216.

In another aspect, the parent is a fragment containing at least 85% ofthe amino acid residues, e.g., at least 90% of the amino acid residuesor at least 95% of the amino acid residues of the mature polypeptide ofa GH61 polypeptide.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215, or thefull-length complements thereof (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179,181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207,209, 211, 213, or 215, or subsequences thereof, as well as thepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216, or fragments thereof, may be used to designnucleic acid probes to identify and clone DNA encoding a parent fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA or cDNA of a cell of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, e.g., at least 25, atleast 35, or at least 70 nucleotides in length. Preferably, the nucleicacid probe is at least 100 nucleotides in length, e.g., at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, or at least 900 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or215, or subsequences thereof, the carrier material is used in a Southernblot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or215; (ii) the mature polypeptide coding sequence thereof; (iii) thefull-length complement thereof; or (iv) a subsequence thereof; undervery low to very high stringency conditions. Molecules to which thenucleic acid probe hybridizes under these conditions can be detectedusing, for example, X-ray film or any other detection means known in theart.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, or 215.

In another aspect, the nucleic acid probe is a polynucleotide thatencodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, or 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, or 216; the mature polypeptide thereof; or afragment thereof.

In another aspect, the nucleic acid probe is SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, or 215.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215 of atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%.

The parent may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial GH61 polypeptide. For example, the parentmay be a Gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces GH61polypeptide, or a Gram-negative bacterial polypeptide such as aCampylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma GH61polypeptide.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis GH61 polypeptide.

In another aspect, the parent is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus GH61 polypeptide.

In another aspect, the parent is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans GH61 polypeptide.

The parent may be a fungal GH61 polypeptide. For example, the parent maybe a yeast GH61 polypeptide such as a Candida, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia GH61 polypeptide; or afilamentous fungal GH61 polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria GH61 polypeptide.

In another aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis GH61 polypeptide.

In another aspect, the parent is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus lentulus,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillusterreus, Chrysosporium inops, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporiumpannicola, Chrysosporium queenslandicum, Chrysosporium tropicum,Chrysosporium zonatum, Fennellia nivea, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium emersonii, Penicillium funiculosum, Penicilliumpinophilum, Penicillium purpurogenum, Phanerochaete chrysosporium,Talaromyces leycettanus, Thermoascus aurantiacus, Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia setosa, Thielavia spededonium, Thielaviasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride GH61 polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a GH61polypeptide variant having cellulolytic enhancing activity, comprising:(a) introducing into a parent GH61 polypeptide a substitution at one ormore (e.g., several) positions corresponding to positions 105, 154, 188,189, 216, and 229 of the mature polypeptide of SEQ ID NO: 30, whereinthe variant has cellulolytic enhancing activity; and optionally (b)recovering the variant. In one aspect, the methods further compriseintroducing into the parent GH61 polypeptide a substitution at one ormore (e.g., several) positions corresponding to positions 111, 152, 155,and 162 of the mature polypeptide of SEQ ID NO: 30, wherein the varianthas cellulolytic enhancing activity. In another aspect, the methodsfurther or even further comprise introducing into the parent GH61polypeptide a substitution at one or more (e.g., several) positionscorresponding to positions 96, 98, 200, 202, and 204 of the maturepolypeptide of SEQ ID NO: 30, wherein the variant has cellulolyticenhancing activity.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Site-saturation mutagenesis systematically replaces a polypeptide codingsequence with sequences encoding all 19 amino acids at one or more(e.g., several) specific positions (Parikh and Matsumura, 2005, J. Mol.Biol. 352: 621-628).

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodingGH61 polypeptide variants of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a GH61 polypeptide variant of the presentinvention operably linked to one or more control sequences that directthe expression of the coding sequence in a suitable host cell underconditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a GH61 polypeptide variant. Manipulation of thepolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides utilizing recombinant DNA methods are wellknown in the art.

The control sequence may be a promoter, a polynucleotide recognized by ahost cell for expression of a polynucleotide encoding a variant of thepresent invention. The promoter contains transcriptional controlsequences that mediate the expression of the GH61 polypeptide variant.The promoter may be any polynucleotide that shows transcriptionalactivity in the host cell including mutant, truncated, and hybridpromoters, and may be obtained from genes encoding extracellular orintracellular polypeptides either homologous or heterologous to the hostcell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding theGH61 polypeptide variant. Any terminator that is functional in the hostcell may be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding theGH61 polypeptide variant. Any leader that is functional in the host cellmay be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the GH61 polypeptidevariant-encoding sequence and, when transcribed, is recognized by thehost cell as a signal to add polyadenosine residues to transcribed mRNA.Any polyadenylation sequence that is functional in the host cell may beused.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus nigerglucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a GH61 polypeptidevariant and directs the variant into the cell's secretory pathway. The5′-end of the coding sequence of the polynucleotide may inherentlycontain a signal peptide coding sequence naturally linked in translationreading frame with the segment of the coding sequence that encodes thevariant. Alternatively, the 5′-end of the coding sequence may contain asignal peptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a GH61 polypeptidevariant. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to an active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Myceliophthora thermophila laccase (WO95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomycescerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the GH61polypeptide variant and the signal peptide sequence is positioned nextto the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the GH61 polypeptide variant relative to the growth of thehost cell. Examples of regulatory sequences are those that causeexpression of the gene to be turned on or off in response to a chemicalor physical stimulus, including the presence of a regulatory compound.Regulatory systems in prokaryotic systems include the lac, tac, and trpoperator systems. In yeast, the ADH2 system or GAL1 system may be used.In filamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding the variantwould be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a GH61 polypeptide variant of thepresent invention, a promoter, and transcriptional and translationalstop signals. The various nucleotide and control sequences may be joinedtogether to produce a recombinant expression vector that may include oneor more convenient restriction sites to allow for insertion orsubstitution of the polynucleotide encoding the variant at such sites.Alternatively, the polynucleotide may be expressed by inserting thepolynucleotide or a nucleic acid construct comprising the polynucleotideinto an appropriate vector for expression. In creating the expressionvector, the coding sequence is located in the vector so that the codingsequence is operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the GH61 polypeptide variant or anyother element of the vector for integration into the genome byhomologous or non-homologous recombination. Alternatively, the vectormay contain additional polynucleotides for directing integration byhomologous recombination into the genome of the host cell at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldcontain a sufficient number of nucleic acids, such as 100 to 10,000 basepairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, whichhave a high degree of sequence identity to the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding polynucleotides.On the other hand, the vector may be integrated into the genome of thehost cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a GH61 polypeptidevariant. An increase in the copy number of the polynucleotide can beobtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the polynucleotide where cells containing amplifiedcopies of the selectable marker gene, and thereby additional copies ofthe polynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a GH61 polypeptide variant of the presentinvention operably linked to one or more control sequences that directthe production of a variant of the present invention. A construct orvector comprising a polynucleotide is introduced into a host cell sothat the construct or vector is maintained as a chromosomal integrant oras a self-replicating extra-chromosomal vector as described earlier. Theterm “host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of aGH61 polypeptide variant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a GH61polypeptide variant, comprising: (a) cultivating a host cell of thepresent invention under conditions suitable for expression of thevariant; and optionally (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the GH61 polypeptide variant using methods known in theart. For example, the cells may be cultivated by shake flaskcultivation, or small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors in a suitable medium and underconditions allowing the variant to be expressed and/or isolated. Thecultivation takes place in a suitable nutrient medium comprising carbonand nitrogen sources and inorganic salts, using procedures known in theart. Suitable media are available from commercial suppliers or may beprepared according to published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the variant is secreted into thenutrient medium, the variant can be recovered directly from the medium.If the variant is not secreted, it can be recovered from cell lysates.

The GH61 polypeptide variant may be detected using methods known in theart that are specific for the variant. These detection methods include,but are not limited to, use of specific antibodies, formation of anenzyme product, or disappearance of an enzyme substrate. For example, anenzyme assay may be used to determine the activity of the variant. See,for example, the assay described in Example 5.

The GH61 polypeptide variant may be recovered using methods known in theart. For example, the variant may be recovered from the nutrient mediumby conventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a whole fermentation broth comprising avariant of the present invention is recovered.

The GH61 polypeptide variant may be purified by a variety of proceduresknown in the art including, but not limited to, chromatography (e.g.,ion exchange, affinity, hydrophobic, chromatofocusing, and sizeexclusion), electrophoretic procedures (e.g., preparative isoelectricfocusing), differential solubility (e.g., ammonium sulfateprecipitation), SDS-PAGE, or extraction (see, e.g., ProteinPurification, Janson and Ryden, editors, VCH Publishers, New York, 1989)to obtain substantially pure variants.

In an alternative aspect, the GH61 polypeptide variant is not recovered,but rather a host cell of the present invention expressing the variantis used as a source of the variant.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a variant of the present invention. Thefermentation broth product further comprises additional ingredients usedin the fermentation process, such as, for example, cells (including, thehost cells containing the gene encoding the polypeptide of the presentinvention which are used to produce the polypeptide of interest), celldebris, biomass, fermentation media and/or fermentation products. Insome embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Thefermentation broth formulations or cell compositions may also compriseone or more (e.g., several) enzymes selected from the group consistingof a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising a variantof the present invention. Preferably, the compositions are enriched insuch a variant. The term “enriched” indicates that the cellulolyticenhancing activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The compositions may comprise a variant of the present invention as themajor enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. The compositions may also comprise one ormore (e.g., several) enzymes selected from the group consisting of ahydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. Thecompositions may be prepared in accordance with methods known in the artand may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the GH61 polypeptide variants having cellulolytic enhancingactivity, or compositions thereof.

The present invention also relates to processes for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a GH61polypeptide variant of the present invention. In one aspect, theprocesses further comprise recovering the degraded or convertedcellulosic material. Soluble products of degradation or conversion ofthe cellulosic material can be separated from insoluble cellulosicmaterial using a method known in the art such as, for example,centrifugation, filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a GH61polypeptide variant of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a GH61 polypeptide variant of the present invention. In oneaspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the processes further compriserecovering the fermentation product from the fermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel, potableethanol, and/or platform chemicals (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom the cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, 0. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the processes of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics?, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbeand Zacchi, 2007, Pretreatment of lignocellulosic materials forefficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresource Technol. 100:10-18; Mosier et al., 2005, Features of promising technologies forpretreatment of lignocellulosic biomass, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosicwastes to improve ethanol and biogas production: A review, Int. J. ofMol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key tounlocking low-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition in the presence of a GH61 polypeptide variant of the presentinvention. The enzymes of the compositions can be added simultaneouslyor sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 80° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 9, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase).

In another aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotherpreferred aspect, the xylanase is a Family 11 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and the GH61 polypeptide variantsdepend on several factors including, but not limited to, the mixture ofcomponent cellulolytic enzymes and/or hemicellulolytic enzymes, thecellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a GH61 polypeptide variant tothe cellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg,about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 toabout 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg,about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25to about 1.0 mg per g of the cellulosic material.

In another aspect, an effective amount of a GH61 polypeptide variant tocellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g,e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75 g, about 0.15to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, orabout 0.05 to about 0.2 g per g of cellulolytic or hemicellulolyticenzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, bacterial, fungal, yeast, plant, or mammalian origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram-positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram-negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), CELLUZYME™(Novozymes A/S), CEREFLO™ (Novozymes A/S), and ULTRAFLO™ (NovozymesA/S), ACCELERASE™ (Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP(Genencor Int.), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™7069 W (Röhm GmbH), FIBREZYME® LDI (Dyadic International, Inc.),FIBREZYME® LBR (Dyadic International, Inc.), or VISCOSTAR® 150 L (DyadicInternational, Inc.). The cellulase enzymes are added in amountseffective from about 0.001 to about 5.0 wt % of solids, e.g., about0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % ofsolids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, anAcidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei CeI7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei CeI5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. Nos. 5,457,046, 5,648,263, and5,686,593.

In the processes of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used as a component of the enzymecomposition.

Examples of GH61 polypeptides having cellulolytic enhancing activityuseful in the processes of the present invention include, but are notlimited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647,WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754),GH61 polypeptides from Penicillium pinophilum (WO 2011/005867),Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397),Thermoascus crustaceus (WO 2011/041504), Aspergillus aculeatus (WO2012/0307990, and Thermomyces lanuginosus (WO 2012/113340). WO2012/146171 discloses GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Humicola insolens.

In one aspect, the GH61 polypeptide variant and GH61 polypeptide havingcellulolytic enhancing activity is used in the presence of a solubleactivating divalent metal cation according to WO 2008/151043, e.g.,manganese or copper.

In another aspect, the GH61 polypeptide variant and GH61 polypeptidehaving cellulolytic enhancing activity is used in the presence of adioxy compound, a bicylic compound, a heterocyclic compound, anitrogen-containing compound, a quinone compound, a sulfur-containingcompound, or a liquor obtained from a pretreated cellulosic materialsuch as pretreated corn stover (PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁵ toabout 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide or variant can be produced by treating alignocellulose or hemicellulose material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and a GH61 polypeptide or variant duringhydrolysis of a cellulosic substrate by a cellulase preparation. Theliquor can be separated from the treated material using a methodstandard in the art, such as filtration, sedimentation, orcentrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹g, about 10⁻³ to about 10⁻¹g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740 L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In another more preferred aspect, the yeast is Saccharomyces cerevisiae.In another more preferred aspect, the yeast is Saccharomyces distaticus.In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Detergent Compositions

The present invention also relates to detergent compositions comprisinga GH61 polypeptide variant of the present invention and a surfactant. AGH61 polypeptide variant of the present invention may be added to andthus become a component of a detergent composition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations. In one aspect, the present invention alsorelates to methods for cleaning or washing a hard surface or laundry,the method comprising contacting the hard surface or the laundry with adetergent composition of the present invention.

In a specific aspect, the present invention provides a detergentadditive comprising a GH61 polypeptide variant of the invention. Thedetergent additive as well as the detergent composition may comprise oneor more (e.g., several) enzymes selected from the group consisting of anamylase, arabinase, cutinase, carbohydrase, cellulase, galactanase,laccase, lipase, mannanase, oxidase, pectinase, peroxidase, protease,and xylanase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include CELLUZYME™, and CAREZYME™(Novozymes A/S), CLAZINASE™, and PURADAX HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metalloprotease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and274.

Preferred commercially available protease enzymes include ALCALASE™SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novozymes A/S),MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ andLIPOLASE ULTRA™ (Novozymes A/S).

Amylases:

Suitable amylases (α and/or β) include those of bacterial or fungalorigin.

Chemically modified or protein engineered mutants are included. Amylasesinclude, for example, α-amylases obtained from Bacillus, e.g., a specialstrain of Bacillus licheniformis, described in more detail in GB1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are DURAMYL™, TERMAMYL™, FUNGAMYL™ andBAN™ (Novozymes A/S), RAPIDASE™ and PURASTAR™ (from GenencorInternational Inc.).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more (e.g., several)enzymes, or by adding a combined additive comprising all of theseenzymes. A detergent additive of the invention, i.e., a separateadditive or a combined additive, can be formulated, for example, as agranulate, liquid, slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste, or a liquid.A liquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more (e.g., several)surfactants, which may be non-ionic including semi-polar and/or anionicand/or cationic and/or zwitterionic. The surfactants are typicallypresent at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more (e.g., several) polymers.Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),poly(vinylimidazole), polycarboxylates such as polyacrylates,maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acidcopolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor. In thedetergent compositions, a GH61 polypeptide variant of the presentinvention having cellulolytic enhancing activity may be added in anamount corresponding to 0.001-100 mg of protein, preferably 0.005-50 mgof protein, more preferably 0.01-25 mg of protein, even more preferably0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and evenmost preferably 0.01-1 mg of protein per liter of wash liquor.

A GH61 polypeptide variant of the present invention having cellulolyticenhancing activity may also be incorporated in the detergentformulations disclosed in WO 97/07202, which is hereby incorporated byreference.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising a polynucleotide of the presentinvention so as to express and produce a GH61 polypeptide variant inrecoverable quantities. The variant may be recovered from the plant orplant part. Alternatively, the plant or plant part containing thevariant may be used as such for improving the quality of a food or feed,e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a variant into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed. For instance, the expression of the gene encoding avariant may be constitutive or inducible, or may be developmental, stageor tissue specific, and the gene product may be targeted to a specifictissue or plant part such as seeds or leaves. Regulatory sequences are,for example, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a variant can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention encompasses not only a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding the variant underconditions conducive for production of the variant; and optionally (b)recovering the variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains Aspergillus oryzae strain PFJO218 (amy⁻, alp⁻, Npl⁻, CPA⁻, KA⁻,pyrG⁻, ku70⁻; U.S. Patent Application 20100221783) was used as anexpression host for the GH61 polypeptide variants.

Aspergillus oryzae strain COLs1300 was also used as an expression hostfor GH61 polypeptide variants. A. niger COLs1300 (amyA, amyB, amyC,alpA, nprA, kusA, niaD, amdS+) was created from A. oryzae PFJ0220 (EP 2147 107 B1) by deleting the promoter and 5′ part of both the nitritereductase (niiA) gene and nitrate reductase (niaD) gene.

Media and Reagents

AMG trace metals solution was composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.H₂O, 3 g of citric acid, and deionized water to 1 liter.

COLs1300 cultivating medium was composed of 100 ml of sucrose medium and1 ml of 1 M urea.

COLs1300 protoplasting solution was composed of 80 mg of GLUCANEX®(Novozymes A/S, Bagsvaerd, Denmark), 0.5 mg/ml of chitinase (SigmaChemical Co., Inc., St. Louis, Mo., USA), 10 ml of 1.2 M MgSO₄, and 100μl of 1 M NaH₂PO₄ pH 5.8.

COVE-N-Gly plates were composed of 50 ml of COVE salt solution, 218 g ofsorbitol, 10 g of glycerol, 2.02 g of KNO₃, 25 g of Noble agar, anddeionized water to 1 liter.

COVE-N-Gly plates with 10 mM uridine were composed of 50 ml of COVE saltsolution, 218 g of sorbitol, 10 g of glycerol, 2.02 g of KNO₃, 25 g ofNoble agar, and deionized water to 1 liter; uridine was then added at aconcentration of 10 mM to individual plates.

COVE salt solution was composed of 26 g of KCl, 26 g of MgSO₄.7H₂O, 76 gof KH₂PO₄, 50 ml of COVE trace elements solution, and deionized water to1 liter.

COVE trace elements solution was composed of 40 mg of Na₂B₄O₇.10H₂O, 0.4g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄. H₂O, 0.8 g ofNa₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofNaCl, and deionized water to 1 liter.

LB+Amp medium was composed of LB medium supplemented with 100 μg ofampicillin per ml.

M400 medium was composed of 50 g of maltodextrin, 2 g of MgSO₄.7H₂O, 2 gof KH₂PO₄, 4 g of citric acid, 8 g of yeast extract, 2 g of urea, 0.5 mlof AMG trace metals solution, 0.5 g of CaCl₂, and deionized water to 1liter; adjusted with NaOH to pH 6. After pH adjustment 0.7 ml ofantifoam was added.

Magnificent Broth was composed of 50 g of Magnificent Broth powder(MacConnell Research Corp. San Diego, Calif., USA) and deionized waterto 1 liter.

MaltV1 medium was composed of 20 g of maltose, 10 g of Bacto Peptone, 1g of yeast extract, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g ofCaCl₂, 0.42 g of MgSO₄.7H₂O, 0.42 ml of Trichoderma trace metalssolution, 0.48 g of citric acid, 19.52 g of2-(N-morpholino)ethanesulfonic acid (MES), and deionized water to 1liter; adjusted with NaOH to pH 5.5.

MDU2BP medium (pH 5.0) was composed of 135 g of maltose, 3 g ofMgSO₄.7H₂O, 3 g of NaCl, 6 g of K₂SO₄, 36 g of KH₂PO₄, 21 g of yeastextract, 6 g of urea, 1.5 ml of AMG trace metals solution, and deionizedwater up to 1 liter.

PEG solution was composed of 6 g of polyethylene glycol 4000 (PEG 4000),100 μl of 1 M Tris pH 7.5, 100 μl of 1 M CaCl₂, and deionized water to10 ml.

Protoplasting cultivation medium was composed of 92 ml of transformationsucrose medium, 2 ml of 1 M uridine, 1 ml of 1 M NaNO₃, and 10 ml of YPmedium.

Protoplasting solution was composed of 15 ml of 1.2 M MgSO₄, 150 μl of 1M NaH₂PO₄ (pH 5.8), 100 mg of GLUCANEX® (Novozymes A/S, Bagsvaerd,Denmark), and 10 mg of chitinase (Sigma Chemical Co., Inc., St. Louis,Mo., USA).

ST solution was composed of 1.5 ml of 2 M sorbitol, 500 μl of 1 M TrispH 7.5, and deionized water to 5 ml.

STC solution was composed of 60 ml of 2 M sorbitol, 1 ml of 1 M Tris pH7.5, 1 ml of 1 M CaCl₂, and deionized water to 100 ml.

Sucrose medium was composed of 20 ml of COVE salt solution, 342 g ofsucrose, and deionized water to 1 liter.

Sucrose agar plate was composed of 20 ml of Trichoderma trace elementsolution, 20 g of Noble agar, 342 g of sucrose, and deionized water to 1liter.

TAE buffer was composed of 40 mM2-amino-2-hydroxymethyl-propane-1,3-diol, 20 mM Glacial acetic acid, and2 mM ethylenediaminetetraacetic acid at pH 8.0.

TBE buffer was composed of 10.8 g of Tris base, 5.5 g of boric acid, and0.74 g of EDTA (pH 8) in deionized water to 1 liter.

TE buffer was composed of 10 mM Tris-0.1 mM EDTA pH 8.

Top agar was composed of 500 ml of sucrose medium, 5 g of low meltingagarose, and 10 ml of 20 mM Tris pH 7.5.

Transformation sucrose medium was composed of 70 ml of 1 M sucrose and20 ml of COVE salt solution.

Trichoderma trace metals solution was composed of 216 g of FeCl₃.6H₂O,58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g ofH₃BO₃, 336 g of citric acid, and deionized water to 1 liter.

2XYT agar plates were composed of 16 g of tryptone, 10 g of yeastextract, 5 g of NaCl, 15 g of Bacto agar, and deionized water to 1liter.

2XYT+Amp agar plates were composed of 2XYT agar supplemented with 100 μgof ampicillin per ml.

YP medium was composed of 10 g of Bacto yeast extract, 20 g of Bactopeptone, and deionized water to 1 liter.

Example 1: Construction of Expression Vectors pMMar44, pMMar49, pMMar45,and pDFng113

Plasmid pMMar44 was constructed as described below for expression of theAspergillus fumigatus GH61B polypeptide, and generation of mutant genelibraries. Additionally, plasmids pMMar49, pMMar45, and pDFng113 wereconstructed as described below for expression of the Aspergillusfumigatus GH61B polypeptide mutant (WO 2012/044835), Penicillium sp.(emersonii) GH61A polypeptide (hereinafter Penicillium emersonii GH61Apolypeptide), and Thermoascus aurantiacus GH61A polypeptide,respectively, and generation of variants.

Plasmid pENI2376 (U.S. Patent Application 20060234340) containing theAMA sequence for autonomous maintenance in Aspergillus was digested withBarn HI and Not I to linearize the plasmid and remove an 8 bp fragment.The digested plasmid was purified using a PCR Purification Kit (QIAGENInc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

The Aspergillus fumigatus GH61B polypeptide coding sequence (FIG. 1; SEQID NO: 29 [genomic DNA sequence] and SEQ ID NO: 30 [deduced amino acidsequence]), mutated Aspergillus fumigatus GH61B polypeptide codingsequence (WO 2012/044835), Penicillium emersonii GH61A polypeptidecoding sequence (SEQ ID NO: 35 [genomic DNA sequence] and SEQ ID NO: 36[deduced amino acid sequence]), and Thermoascus aurantiacus GH61Apolypeptide coding sequence (SEQ ID NO: 13 [genomic DNA sequence] andSEQ ID NO: 14 [deduced amino acid sequence]) were amplified from sourceplasmids described below using the primers shown in Table 1. Boldletters represent coding sequence. The remaining sequences arehomologous to insertion sites of pENI2376 for expression of the GH61polypeptide coding sequences.

TABLE 1 GH61 Polypeptide Source origin Template Plasmid Primer IDPrimer Sequence Aspergillus pAG43 (WO pMMar44 AspfuGH61BpCACAACTGGGGATCCATGACT fumigatus 2010/138754) ENI2376F_2TTGTCCAAGATCACTTCCA GH61B (SEQ ID NO: 217) AspfuGH61BpGGCCTCCGCGGCCGCTTAAG ENI2376R_2 CGTTGAACAGTGCAGGACCA (SEQ ID NO: 218)Mutated pTH230 (WO pMMar49 AfumGH61SD CACAACpATGGGGATCCATGACTAspergillus 2012/044835) MB3pENI3376F TTGCCAAGATCACTTCCA fumigatus(SEQ ID NO: 219) GH61B AfumGH61SD GGCCTCCGCGGCCGCTTAAG MB3pENI3376RCGTTGAACAGTGCAGGACCA (SEQ ID NO: 220) Penicillium pDM286 pMMar45PenemGH61pE CACAACTGGGGATCCATGCTG emersonii NI2376F TCTTCGACGACTCGCACCCGH61A (SEQ ID NO: 221) PenemGH61pE GGCCTCCGCGGCCGCCTAGA NI2376RACGTCGGCTCAGGCGGCCCC (SEQ ID NO: 222) Thermoascus pDZA2 pDFng113TaGH61aBaM CTGGGGATCCATGTCCTTTTC aurantiacus (WO HltagFCAAGAT (SEQ ID NO: 223) GH61A 2005/074656) TaGH61aNcoltCTCCGCGGCCGCTTAACCAGT agR ATACAGAG (SEQ ID NO: 224)

Construction of plasmid pMMar44 containing the Aspergillus fumigatusGH61B polypeptide coding sequence is described below. The Aspergillusfumigatus GH61B polypeptide coding sequence was amplified from plasmidpAG43 (WO 2010/138754) using the primers shown in Table 1 with overhangsdesigned for cloning into plasmid pENI2376.

Fifty picomoles of each of the primers listed in Table 1 were used in aPCR reaction composed of 90 ng of pAG43, 1× ADVANTAGE® 2 PCR Buffer(Clontech Laboratories, Inc., Mountain View, Calif., USA), 1 μl of ablend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 1× ADVANTAGE® 2DNA Polymerase Mix (Clontech Laboratories, Inc., Mountain View, Calif.,USA), in a final volume of 50 μl. The amplification was performed usingan EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury,N.Y., USA) programmed for 1 cycle at 95° C. for 1 minute; 30 cycles eachat 95° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1minute; and a final elongation at 72° C. for 10 minutes. The heat blockthen went to a 4° C. soak cycle.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 862 bp PCR product band wasexcised from the gel and extracted using a QIAQUICK® Gel Extraction Kit(QIAGEN Inc., Valencia, Calif., USA).

The homologous ends of the 862 bp PCR product and the digested pENI2376were joined together using an IN-FUSION™ ADVANTAGE® PCR Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA). A total of 63ng of the 862 bp PCR product and 200 ng of the Barn HI/Not I digestedpENI2376 were used in a reaction composed of 4 μl of 5× IN-FUSION™reaction buffer (Clontech Laboratories, Inc., Mountain View, Calif.,USA) and 2 μl of IN-FUSION™ enzyme (Clontech Laboratories, Inc.,Mountain View, Calif., USA), in a final volume of 20 μl. The reactionwas incubated for 15 minutes at 37° C., followed by 15 minutes at 50°C., and then placed on ice. The reaction volume was increased to 100 μlwith TE buffer and 2 μl of the reaction were transformed into E. coliXL10-GOLD® Super Competent Cells (Stratagene, La Jolla, Calif., USA)according to the manufacturer's instructions. E. coli transformants wereselected on 2XYT+Amp agar plates. Plasmid DNA from several of theresulting E. coli transformants was prepared using a BIOROBOT® 9600(QIAGEN Inc., Valencia, Calif., USA). The Aspergillus fumigatus GH61Bpolypeptide coding sequence insert was confirmed by DNA sequencing witha Model 377 XL Automated DNA Sequencer (Applied Biosystems Inc., FosterCity, Calif., USA) using dye-terminator chemistry (Giesecke et al.,1992, J. Virol. Methods 38: 47-60). Sequencing primers used forverification of the gene insert and sequence are shown below.

Primer 996271: (SEQ ID NO: 225) ACTCAATTTACCTCTATCCACACTTPrimer pALLO2 3′: (SEQ ID NO: 226) GAATTGTGAGCGGATAACAATTTCA

A plasmid containing the correct A. fumigatus GH61B polypeptide codingsequence was selected and designated pMMar44 (FIG. 2).

Construction of plasmid pMMar49 containing eight base-pair changesresulted in four amino acid mutations of the Aspergillus fumigatus GH61Bpolypeptide (WO 2012/044835) is described below. The mutated Aspergillusfumigatus GH61B polypeptide coding sequence (WO 2012/044835) wasamplified from plasmid pTH230 using the primers shown in Table 1 withoverhangs designed for cloning into plasmid pENI2376.

Fifty picomoles of each of the primers listed in Table 1 were used in aPCR reaction composed of 100 ng of pTH230, 1× ADVANTAGE® 2 PCR Buffer(Clontech Laboratories, Inc., Mountain View, Calif., USA), 1 μl of ablend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 1× ADVANTAGE® 2DNA Polymerase Mix (Clontech Laboratories, Inc., Mountain View, Calif.,USA), in a final volume of 50 μl. The amplification was performed usingan EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 1minute; 30 cycles each at 95° C. for 30 seconds, 60° C. for 30 seconds,and 72° C. for 1 minute; and a final elongation at 72° C. for 7 minutes.The heat block then went to a 4° C. soak cycle.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 862 bp PCR product band wasexcised from the gel and extracted using a QIAQUICK® Gel Extraction Kit.

The homologous ends of the 862 bp PCR product and the digested pENI2376were joined together using an IN-FUSION™ ADVANTAGE® PCR Cloning Kit. Atotal of 90 ng of the 862 bp PCR product and 220 ng of the Barn HI/Not Idigested pENI2376 were used in a reaction composed of 4 μl of 5×IN-FUSION™ reaction buffer and 2 μl of IN-FUSION™ enzyme in a finalvolume of 20 μl. The reaction was incubated for 15 minutes at 37° C.,followed by 15 minutes at 50° C., and then placed on ice. The reactionvolume was increased to 100 μl with TE buffer and 2 μl of the reactionwere transformed into E. coli XL10-GOLD® Super Competent Cells accordingto the manufacturer's instructions. E. coli transformants were selectedon 2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.coli transformants was prepared using a BIOROBOT® 9600. The mutatedAspergillus fumigatus GH61B polypeptide coding sequence insert wasconfirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencerusing dye-terminator chemistry (Giesecke et al., 1992, supra). Thesequencing primers 996271 and pALLO2 3′ were used for verification ofthe gene insert and sequence.

A plasmid containing the correct mutated A. fumigatus GH61B polypeptidecoding sequence was selected and designated pMMar49 (FIG. 3).

Construction of plasmid pMMar45 containing the Penicillium emersoniiGH61A polypeptide coding sequence is described below. The Penicilliumemersonii GH61A polypeptide coding sequence was amplified from plasmidpDM286 containing the Penicillium emersonii GH61A polypeptide codingsequence using the primers shown in Table 1 with overhangs designed forcloning into plasmid pENI2376.

Plasmid pDM286 was constructed according to the following protocol. TheP. emersonii GH61A polypeptide gene was amplified from plasmidpGH61D23Y4 (WO 2011/041397) using PHUSION™ High-Fidelity Hot Start DNAPolymerase (Finnzymes Oy, Espoo, Finland) and gene-specific forward andreverse primers shown below. The region in italics represents vectorhomology to the site of insertion.

Forward primer: (SEQ ID NO: 227)5′-CGGACTGCGCACCATGCTGTCTTCGACGACTCGCAC-3′ Reverse primer:(SEQ ID NO: 228) 5′-TCGCCACGGAGCTTATCGACTTCTTCTAGAACGTC-3′

The amplification reaction contained 30 ng of plasmid pGH61D23Y4, 50pmoles of each of the primers listed above, 1 μl of a 10 mM blend ofdATP, dTTP, dGTP, and dCTP, 1× PHUSION™ High-Fidelity Hot Start DNAPolymerase buffer (Finnzymes Oy, Espoo, Finland) and 1 unit of PHUSION™High-Fidelity Hot Start DNA Polymerase buffer (Finnzymes Oy, Espoo,Finland) in a final volume of 50 μl.

The amplification reaction was incubated in an EPPENDORF® MASTERCYCLER®5333 epgradient S (Eppendorf Scientific, Inc., Westbury, N.Y., USA)programmed for 1 cycle at 98° C. for 30 seconds; 35 cycles each at 98°C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds, and1 cycle at 72° C. for 10 minutes.

PCR products were separated by 1% agarose gel electrophoresis using TAEbuffer. A 0.87 kb fragment was excised from the gel and extracted usinga NUCLEOSPIN® Extract II Kit (Macherey-Nagel, Inc., Bethlehem, Pa., USA)according to the manufacturer's protocol.

Plasmid pMJ09 (US 2005/0214920 A1) was digested with Nco I and Pac I,and after digestion, the digested vector was isolated by 1.0% agarosegel electrophoresis using TBE buffer where an approximately 7.1 kbfragment was excised from the gel and extracted using a QIAQUICK® GelExtraction Kit. The 0.87 kb PCR product was inserted into Nco I/PacI-digested pMJ09 using an IN-FUSION™ ADVANTAGE® PCR Cloning Kitaccording to the manufacturer's protocol. The IN-FUSION™ reaction wascomposed of 1× IN-FUSION™ Reaction buffer, 180 ng of Not I/Pac Idigested plasmid pMJ09, 108 ng of the 0.87 kb PCR product, and 1 μl ofIN-FUSION™ Enzyme in a 10 μl reaction volume. The reaction was incubatedfor 15 minutes at 37° C. and then for 15 minutes at 50° C. To thereaction 40 μl of TE were added and 2 μl were used to transform ONESHOT® TOP10 competent cells (Invitrogen, Carlsbad, Calif., USA)according to the manufacturer's protocol. Transformants were screened bysequencing and one clone containing the insert with no PCR errors wasidentified and designated plasmid pDM286. Plasmid pDM286 can be digestedwith Pme I to generate an approximately 5.4 kb fragment for T. reeseitransformation. This 5.4 kb fragment contains the expression cassette[T. reesei Cel7A cellobiohydrolase (CBHI) promoter, P. emersoniiglycosyl hydrolase 61A (GH61A) gene, T. reesei Cel7A cellobiohydrolase(CBHI) terminator], and Aspergillus nidulans acetamidase (amdS)gene.

For construction of pMMar45, 50 picomoles of each of the primers listedin Table 1 were used in a PCR reaction composed of 120 ng of pDM286, 1×EXPAND® PCR Buffer (Roche Diagnostics, Inc., Indianapolis, Ind., USA), 1μl of a blend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 1×EXPAND® DNA Polymerase Mix (Roche Diagnostics, Inc., Indianapolis, Ind.,USA), in a final volume of 50 μl. The amplification was performed usingan EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 1minute; 30 cycles each at 95° C. for 30 seconds, 60° C. for 30 seconds,and 72° C. for 1 minute; and a final elongation at 72° C. for 10minutes. The heat block then went to a 4° C. soak cycle.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 762 bp PCR product band wasexcised from the gel and extracted using a QIAQUICK® Gel Extraction Kit.

The homologous ends of the 762 bp PCR product and the Barn HI/Not Idigested pENI2376 were joined together using an IN-FUSION™ ADVANTAGE®PCR Cloning Kit. A total of 90 ng of the 762 bp PCR product and 200 ngof the digested pENI2376 were used in a reaction composed of 4 μl of 5×IN-FUSION™ reaction buffer and 2 μl of IN-FUSION™ enzyme, in a finalvolume of 20 μl. The reaction was incubated for 15 minutes at 37° C.,followed by 15 minutes at 50° C., and then placed on ice. The reactionvolume was increased to 100 μl with TE buffer and 2 μl of the reactionwere transformed into E. coli XL10-GOLD® Super Competent Cells accordingto the manufacturer's instructions. E. coli transformants were selectedon 2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.coli transformants was prepared using a BIOROBOT® 9600. The P. emersoniiGH61A polypeptide coding sequence insert was confirmed by DNA sequencingwith a Model 377 XL Automated DNA Sequencer using dye-terminatorchemistry (Giesecke et al., 1992, supra). The sequencing primers 996271and pALLO2 3′ were used for verification of the gene insert andsequence.

A plasmid containing the correct P. emersonii GH61A polypeptide codingsequence was selected and designated pMMar45 (FIG. 4).

Construction of plasmid pDFng113 containing the Thermoascus aurantiacusGH61A polypeptide coding sequence is described below. The Thermoascusaurantiacus GH61A polypeptide coding sequence was amplified from plasmidpDZA2 (WO 2005/074656) using the primers shown in Table 1 with overhangsdesigned for cloning into plasmid pENI2376.

Fifty picomoles of each of the primers listed in Table 1 were used in aPCR reaction composed of 100 ng of pDZA2, 1× EXPAND® PCR Buffer, 1 μl ofa blend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 1× EXPAND® DNAPolymerase Mix, in a final volume of 50 μl. The amplification wasperformed using an EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycleat 94° C. for 2 minutes; 30 cycles each at 94° C. for 15 seconds, 59.9°C. for 30 seconds, and 72° C. for 1 minute; and a final elongation at72° C. for 7 minutes. The heat block then went to a 4° C. soak cycle.

The reaction product was isolated by 1.0% agarose gel electrophoresisusing TBE buffer where an approximately 822 bp PCR product band wasexcised from the gel and extracted using a QIAQUICK® Gel Extraction Kit.

The homologous ends of the 822 bp PCR product and the Barn HI/Not Idigested pENI2376 were joined together using an IN-FUSION™ ADVANTAGE®PCR Cloning Kit. A total of 37 ng of the 799 bp PCR product and 200 ngof the digested pENI2376 were used in a reaction composed of 4 μl of 5×IN-FUSION™ reaction buffer and 2 μl of IN-FUSION™ enzyme in a finalvolume of 20 μl. The reaction was incubated for 15 minutes at 37° C.,followed by 15 minutes at 50° C., and then placed on ice. The reactionvolume was increased to 50 μl with TE buffer and 2 μl of the reactionwere transformed into E. coli XL10-GOLD® Ultra Competent Cells accordingto the manufacturer's instructions. E. coli transformants were selectedon 2XYT+Amp agar plates. Plasmid DNA from several of the resulting E.coli transformants was prepared using a BIOROBOT® 9600. The T.aurantiacus GH61A polypeptide coding sequence insert was confirmed byDNA sequencing with a Model 377 XL Automated DNA Sequencer usingdye-terminator chemistry (Giesecke et al., 1992, supra). The sequencingprimers 996271 and pALLO2 3′ were used for verification of the geneinsert and sequence.

A plasmid containing the correct T. aurantiacus GH61A polypeptide codingsequence was selected and designated pDFng113 (FIG. 5).

Example 2: Construction of an Aspergillus fumigatus GH61B PolypeptideSite Saturation Library

A site saturation library of the Aspergillus fumigatus GH61B polypeptidecoding sequence was synthesized by GeneArt AG (Regensburg, Germany). Anaverage of 16.8 mutations per position was synthesized for a total of165 residues, excluding the most conserved residues, resulting in atotal of 2768 mutants. E. coli DH10B (Invitrogen, Carlsbad, Calif., USA)strains containing mutant plasmids with known mutations were arrayed in96 well plates as 50 μl glycerol stocks, and stored at −80° C.

DNA was generated from a thawed GeneArt plate by using a sterile 96 wellreplicator to stamp the GeneArt plate onto a 2XYT+Amp agar plate. Theagar plate was incubated overnight at 37° C. Resulting colonies from theagar plate were used to inoculate a 96 deep well block with each wellcontaining 1 ml of Magnificent broth supplemented with 400 μg ofampicillin per ml. The block was covered with an airpore breathable lidand then incubated in a humidified box at 37° C. overnight at 350 rpm.The block was centrifuged at 1100×g for 10 minutes and the supernatantdiscarded. Plasmids were extracted from the cell pellets using aBIOROBOT® 9600.

Example 3: Expression of the A. fumigatus GH61B, P. emersonii GH61A, andT. aurantiacus GH61A Polypeptide Variants in Aspergillus oryzae PFJO218

Aspergillus oryzae PFJO218 was inoculated onto a COVE-N-Gly plate with10 mM uridine and incubated at 34° C. until confluent. Spores werecollected from the plate by washing with 8 ml of 0.01% TWEEN® 20. One mlof the spore suspension was used to inoculate 103 ml of theProtoplasting cultivation medium in a 500 ml polycarbonate shake flask.The shake flask was incubated at 30° C. with agitation at 180 rpm for17-20 hours. Mycelia were filtered through a funnel lined withMIRACLOTH® (Calbiochem, La Jolla, Calif., USA) and washed with 200 ml of0.6 M MgSO₄. Washed mycelia were resuspended in 15 ml of Protoplastingsolution in a 125 ml sterile polycarbonate shake flask and incubated onice for 5 minutes. One ml of a solution of 12 mg of bovine serum albuminper ml of deionized water was added to the shake flask and the shakeflask was then incubated at 37° C. with mixing at 70 rpm for 1-3 hoursuntil protoplasting was complete. The mycelia/protoplast mixture wasfiltered through a funnel lined with MIRACLOTH® into a 50 ml conicaltube and overlayed with 5 ml of ST. The 50 ml conical tube wascentrifuged at 1050×g for 15 minutes with slowacceleration/deceleration. After centrifugation, the liquid wasseparated into 3 phases. The interphase which contained the protoplastswas transferred to a new 50 ml conical tube. Two volumes of STC wereadded to the protoplasts followed by a brief centrifugation at 1050×gfor 5 minutes. The supernatant was discarded. The protoplasts werewashed twice with 20 ml of STC with resuspension of the protoplastpellet, centrifugation at 1050×g for 10 minutes, and decanting of thesupernatant each time. After the final decanting, the protoplast pelletwas resuspended in STC at a concentration of 1×10⁸/ml. Protoplasts werefrozen at −80° C. until transformation.

A 1.3 μl volume of each mutant plasmid was used to transform 3.5 μl ofA. oryzae PFJO218 protoplasts with 3.5 μl of PEG solution per well in a24 well plate. Plasmid pMMar44, pMMar45, or pDFng113 (Table 1) was alsotransformed as above into A. oryzae PFJO218 protoplasts to provide brothcomprising the A. fumigatus, P. emersonii, or T. aurantiacus wild-typeGH61 polypeptides. The 24 well plate was incubated at 37° C. stationaryfor 30 minutes followed by addition of 28.6 μl of Transformation sucrosemedium containing 10 mM NaNO₃ and 14.3 μl of STC. The 24 well plate wasthen placed in a humidified box at 37° C. stationary for 7 days. On day7, 1 ml of MaltV1 medium was added to each well. The plate was returnedto the humidified box at 39° C. stationary and incubated for anadditional 5 days. At least 550 μl of broth for each variant or thewild-type A. fumigatus, P. emersonii, or T. aurantiacus GH61 polypeptidewere harvested using a pipette to remove the mycelia mat and aspiratethe liquid, for assay using PASC as a substrate. Mutant plasmidsresulting in variants with improved thermostability using a PASC assay(Example 5) were transformed again and retested using the protocolsdescribed above.

Some of the variants were spore-purified for further characterization.After a 7 day incubation of the transformation and prior to the additionof 1 ml of MaltV1 expression medium, a loop was swiped over the initialgrowth from the transformation to collect spores in the well. The sporeswere then streaked onto a COVE-N-Gly plate and incubated at 37° C. forapproximately 36 hours. Single individual transformants were excisedfrom the plate and transferred onto fresh COVE-N-Gly plates. The plateswere stored at 34° C. until confluent. Once confluent, a loop dipped in0.01% TWEEN® 20 was swiped over the spores which was then used toinoculate a 24 well plate with each well containing 1 ml of MaltV1expression medium. The 24 well plate was placed in a humidified box at39° C. Samples were harvested on the fifth day by removing the myceliamat and pipetting up the broth.

Example 4: Preparation of Aspergillus fumigatus Beta-Glucosidase

Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ ID NO: 243[DNA sequence] and SEQ ID NO: 244 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2005/047499 using Aspergillusoryzae as a host.

The filtered broth was adjusted to pH 8.0 with 20% sodium acetate, whichmade the solution turbid. To remove the turbidity, the solution wascentrifuged at 20,000×g for 20 minutes, and the supernatant was filteredthrough a 0.2 μm filtration unit (Nalgene, Rochester, N.Y., USA). Thefiltrate was diluted with deionized water to reach the same conductivityas 50 mM Tris/HCl pH 8.0. The adjusted enzyme solution was applied to aQ SEPHAROSE® Fast Flow column (GE Healthcare, Piscataway, N.J., USA)equilibrated with 50 mM Tris-HCl pH 8.0 and eluted with a lineargradient from 0 to 500 mM sodium chloride. Fractions were pooled andtreated with 1% (w/v) activated charcoal to remove color from thebeta-glucosidase pool. The charcoal was removed by filtration of thesuspension through a 0.2 μm filtration unit (Nalgene, Rochester, N.Y.,USA). The filtrate was adjusted to pH 5.0 with 20% acetic acid anddiluted 10 times with deionized water. The adjusted filtrate was appliedto a SP SEPHAROSE® Fast Flow column (GE Healthcare, Piscataway, N.J.,USA) equilibrated with 10 mM succinic acid pH 5.0 and eluted with alinear gradient from 0 to 500 mM sodium chloride. Protein concentrationwas determined using a Microplate BCA™ Protein Assay Kit (Thermo FischerScientific, Waltham, Mass., USA) in which bovine serum albumin was usedas a protein standard.

Example 5: Screening of Aspergillus fumigatus GH61B Polypeptide VariantLibraries

Using a BIOMEK® FX Laboratory Automation Workstation (Beckman Coulter,Fullerton, Calif., USA) with a DYAD® Thermal Cycler (Bio-RadLaboratories, Inc., Richmond, Calif., USA), 80 μl of each broth samplefrom the library plates of the Aspergillus fumigatus GH61B variants andparent (wild-type) polypeptide grown in MaltV1 medium (Example 3) weremixed with 20 μl of 1 M sodium acetate-10 mM MnSO₄ pH 5.0 buffer. Thesamples were then heat challenged at 62° C., 65° C., and 68° C. for 20minutes and compared to ambient temperature controls. After the heatchallenge, the broth samples were diluted 1.25, 2.5, 6.25, and15.625-fold in 2 mM MnSO₄-200 mM sodium acetate pH 5 and 12.5 μl of thedilutions were then transferred to 384-well polypropylene assay platescontaining 25 μl of 1% phosphoric acid swollen cellulose (PASC) and 12.5μl of a cofactor solution (400 mM sodium acetate pH 5, 4 mM MnSO₄, 0.4%gallic acid, 0.1 mg/ml of Aspergillus fumigatus beta-glucosidase, and0.04% TRITON® X100). The plates were heat-sealed using an ALPS-300™(Abgene, Epsom, United Kingdom) with a plastic sheet and incubated at40° C. for 4 days.

Background glucose concentration of the buffer-treated broth samples wasdetermined prior to incubation by performing a glucose assay using thefollowing reagents per liter: 0.9951 g of ATP, 0.5176 g of NAD, 0.5511 gof MgSO₄.7H₂O, 20.9 g of MOPS, 1000 units of hexokinase, 1000 units ofglucose-6-phosphate dehydrogenase, and 0.01% TRITON® X-100, pH 7.5. TheBIOMEK® FX Laboratory Automation Workstation was used for this assay.Four 2-fold serial dilutions were performed in 384-well polystyreneplates using water as diluent. Five μl of the dilutions were added to anew 384-well polystyrene plate, followed by addition of 60 μl of theabove reagents. The plate was incubated at ambient temperature (22°C.±2° C.) for 30 to 45 minutes. Relative fluorescent units (RFU) weredetermined using a DTX 880 plate reader (Beckman Coulter, Fullerton,Calif., USA) with excitation at 360 nm and emission at 465 nm andcompared to glucose standards (1 mg/ml and 0.125 mg/ml) diluted in thesame plate as the samples. At the end of four days, the 40° C. incubatedPASC plates were analyzed for glucose concentration using the glucoseassay described above. Any background glucose was subtracted from theappropriate samples and then residual activity was calculated bycomparing the glucose released in the PASC assay of the ambient sampletreatment to the glucose released in the PASC assay of the heatchallenge sample treatment. Only data that fits in the linear part ofthe curve (defined as less than or equal to 1 mg/ml glucose produced inan assay containing 5 mg/ml PASC) was used in the calculation. Theformula for calculating the residual activity of the heat treatment wasas follows: (mg/ml glucose produced for heat treated sample/mg/mlglucose produced for ambient treated sample)×100%. Improved variantswere those having a higher % residual activity as compared to wild-typeA. fumigatus GH61A polypeptide broth from MaltV1 medium in at least oneheat treatment condition. MICROSOFT® EXCEL® (Microsoft Corporation,Redmond, Wash., USA) was used for all calculations.

Example 6: Thermostability of Aspergillus fumigatus GH61B PolypeptideVariants Measured by Residual Activity after Heat Treatment

Based on the residual activity ratios as described in Example 5,screening of libraries constructed in the previous Examples generatedthe results listed in Tables 2 and 3.

Table 2 shows average % Residual Activity (from 3-5 samples of eachvariant and the wild type control) after treatment at 62, 65, or 68° C.The parent Aspergillus fumigatus GH61B polypeptide showed decreasedresidual activity of 70%, 45%, and 22% when the temperature wasincreased from 62° C. to 65° C. to 68° C., respectively. The increase inthermostability of the Aspergillus fumigatus GH61B polypeptide variantsranged from 1.03- to 1.1-fold increase at 62° C., 1.09- to 1.4-foldincrease at 65° C., and 1.5- to 2.5-fold increase at 68° C. treatmentcompared to the wild-type A. fumigatus GH61 polypeptide. The resultsshowed that improvements were most significant at 68° C. treatment.

TABLE 2 Variants with improved thermostability at 62° C., 65° C., and68° C. treatment Avg % Res. Avg % Res. Avg % Res. Act. 62° C. StandardAct. 65° C. Standard Act. 68° C. Standard Variant treatment Deviationtreatment Deviation treatment Deviation Parent (Wild-Type) 70% 9% 45% 4%22% 6% E105K 72% 6% 54% 3% 39% 4% E105P 80% 11%  54% 3% 33% 4% E154L 78%8% 64% 3% 47% 4% G188A 77% 7% 62% 8% 55% 8% G188W 75% 13%  63% 8% 46% 7%N189K 73% 1% 61% 6% 46% 8% A216L 75% 8% 62% 4% 42% 3% A216Y 77% 6% 59%3% 42% 3% K229H 76% 13%  59% 2% 40% 3% K229I 76% 3% 56% 2% 36% 6% K229W66% 8% 49% 9% 43% 10%  K229Y 75% 3% 54% 4% 31% 2%

Table 3 shows average % Residual Activity (from 3-5 samples of eachvariant and 110 samples of the wild type control) after treatment at 62°C., 65° C., or 68° C. The parent Aspergillus fumigatus GH61B polypeptideshowed decreased residual activity of 56%, 35%, and 12% when thetemperature was increased from 62° C. to 65° C. to 68° C., respectively.The increase in thermostability of the Aspergillus fumigatus GH61Bpolypeptide variants ranged from 1.02-fold to 1.2-fold increase at 62°C., 1.14-fold to 1.6-fold increase at 65° C., and 2.08-fold to 3.25-foldincrease at 68° C. treatment compared to the wild-type A. fumigatus GH61polypeptide. The results showed that improvements were most significantat 68° C. treatment.

TABLE 3 Aspergillus fumigatus GH61B polypeptide variants with improvedthermostability at 62° C., 65° C., and 68° C. treatment Avg % Res. Avg %Res. Avg % Res. Act. 62° C. Standard Act. 65° C. Standard Act. 68° C.Standard Variant treatment Deviation treatment Deviation treatmentDeviation Parent (Wild-Type) 56% 13% 35% 16% 12% 10% E105K 61% 24% 50%23% 38% 21% E105P 57% 23% 44% 21% 32% 21% E154I 68% 13% 57% 16% 34%  9%E154L 56% 26% 41% 23% 25% 20% G188F 56% 21% 45% 22% 33% 27% G188M 61%13% 51% 13% 34% 15% G188A 57% 24% 44% 25% 39% 24% G188W 52% 24% 40% 25%31% 26% N189H 60% 18% 44% 19% 26% 18% N189K 51% 23% 41% 24% 30% 21%A216Y 56% 27% 43% 27% 32% 23% A216L 56% 24% 46% 25% 33% 21% K229W 51%22% 41% 21% 39% 26% K229H 58% 24% 46% 22% 31% 21% K229I 58% 25% 45% 24%30% 21% K229Y 55% 23% 43% 22% 28% 18%

Example 7: Thermostability of Aspergillus fumigatus GH61B CombinatorialVariants

Four variants of the Aspergillus fumigatus GH61B polypeptide wereconstructed by performing site-directed mutagenesis on pMMar49(Example 1) using a QUIKCHANGE® II XL Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif., USA). Two mutagenic primers were designedfor each construct to insert the desired mutation. 125 ng of each primer(Table 4) was used in a PCR reaction containing approximately 25 ng oftemplate plasmid, lx QUIKCHANGE® reaction buffer, 3 μl of QUIKSOLUTION®,1 μl of XL dNTP mix, and 1 μl of 2.5 U/μl Pfu Ultra enzyme in a finalvolume of 50 μl. An EPPENDORF® MASTERCYCLER® thermocycler was used withthe following settings: 95° C. hot start, one cycle at 95° C. for 1minute; 18 cycles each at 95° C. for 50 seconds, 60° C. for 50 seconds,and 68° C. for 10 minutes; and 4° C. hold. One microliter of Dpn I wasdirectly added to the amplification reaction and incubated at 37° C. for1 hour. A 2 μl volume of the Dpnl digested reaction was used totransform E. coli XL10-Gold Ultracompetent Cells (Stratagene, La Jolla,Calif.) according to the manufacturer's instructions. E. colitransformants were selected on 2XYT+Amp agar plates. One of the cloneswith the desired mutation was designated as each plasmid listed below.

Mutations G188A, G188W, K229W, and N189K were added individually on topof the A. fumigatus GH61B polypeptide variant containing mutationsL111V, D152S, M155L, and A162W (pMMar49, Example 1), resulting inpLSBF09-1, pLSBF09-2, pLSBF09-3, and pLSBF09-4, respectively. A summaryof the oligos used for the site-directed mutagenesis reactions are shownbelow in Table 4.

Three additional variants of Aspergillus fumigatus GH61B wereconstructed by performing site-directed mutagenesis on pLSBF09-3 using aQUIKCHANGE® II XL Site-Directed Mutagenesis Kit as described above.Mutations G188F, N 189K, and A216Y were individually added as described,resulting in pDFNG146, pDFNG147, and pLSBF21. A summary of the oligosused for the site-directed mutagenesis reactions are shown below inTable 4.

The seven variant plasmids above were prepared using a BIOROBOT® 9600.The variant plasmid constructs were then sequenced using a 3130xlGenetic Analyzer (Applied Biosystems, Foster City, Calif., USA) toverify the changes.

TABLE 4 Plasmid Mutation Oligo ID # Sequence pLSBF09-1 L111V, 615626ATCATCGCCCTTCACTCTGCGGCCAACCTGA D152S, ACGGCGCGCAGAAC (SEQ ID NO: 229)M155L, 615630 GTTCTGCGCGCCGTTCAGGTTGGCCGCAGA A162W,GTGAAGGGCGATGAT (SEQ ID NO: 230) G188A pLSBF09-2 L111V, 615627ATCATCGCCCTTCACTCTGCGTGGAACCTGA D152S, ACGGCGCGCAGAAC (SEQ ID NO: 231)M155L, 615631 GTTCTGCGCGCCGTTCAGGTTCCACGCAGA A162W,GTGAAGGGCGATGAT (SEQ ID NO: 232) G188W pLSBF09-3 L111V, 615628ACAAGAATACTGATCCTGGCATCTGGTTTGA D152S,CATCTACTCGGATCTGAG (SEQ ID NO: 233) M155L, 615632CTCAGATCCGAGTAGATGTCAAACCAGATGC A162W,CAGGATCAGTATTCTTGT (SEQ ID NO: 234) K229W pLSBF09-4 L111V, 615629TCGCCCTTCACTCTGCGGGTAAGCTGAACGG D152S, CGCGCAGAACTAC (SEQ ID NO: 235)M155L, 615633 GTAGTTCTGCGCGCCGTTCAGCTTACCCGCA A162W,GAGTGAAGGGCGA (SEQ ID NO: 236) N189K pDFng146 L111V, 1200378ATCATCGCCCTTCACTCTGCGTTTAACCTGAA D152S, CGGCGCGCAGAAC (SEQ ID NO: 237)M155L, 1200379 GTTCTGCGCGCCGTTCAGGTTAAACGCAGAG A162W,TGAAGGGCGATGAT (SEQ ID NO: 238) G188F, K229W pDFng147 L111V, 1200380TCGCCCTTCACTCTGCGGGTAAGCTGAACGG D152S, CGCGCAGAACTAC (SEQ ID NO: 239)M155L, 1200381 GTAGTTCTGCGCGCCGTTCAGCTTACCCGCA A162W,GAGTGAAGGGCGA (SEQ ID NO: 240) N189K K229W pLSBF21 L111V, 1200277GTGCTCAGGGATCTGGCACCTACGGCACGT D152S, CCCTGTACAAGAATA (SEQ ID NO: 241)M155L, 1200278 TATTCTTGTACAGGGACGTGCCGTAGGTGCC A162W,AGATCCCTGAGCAC (SEQ ID NO: 242) A216Y K229W

Based on the residual activity ratios determined according to Example 5,screening of libraries constructed in the previous Examples generatedthe results listed in Table 5. Table 5 shows an average % ResidualActivity (from 1-14 samples each for the combinatorial variants and 23samples of the wild type) after treatment at 65° C., 68° C., or 72° C.

The parent Aspergillus fumigatus GH61 B polypeptide showed decreasedresidual activity of 33%, 3%, and 1% when the temperature of treatmentwas increased from 65° C. to 68° C. to 72° C., respectively. Theincrease in thermostability of the Aspergillus fumigatus GH61Bpolypeptide combinatorial variants ranged from 1.66-fold to 2.42-foldincrease at 65° C., 14.36-fold to 19.57-fold increase at 68° C., and31.45-fold to 80.07-fold increase at 72° C. compared to the wild-type A.fumigatus GH61 polypeptide. The results showed that improvements weremost significant at 72° C. treatment.

TABLE 5 Aspergillus fumigatus GH61B polypeptide variants with improvedthermostability at 65° C., 68° C., and 72° C. treatment Avg % Res.Standard Avg % Res. Standard Avg % Res. Standard Mutations Act. 65° C.Deviation Act. 68° C. Deviation Act. 72° C. Deviation L111V, D152S,M155L, 55% 10% 56% 2% 51% 7% A162W, G188F, K229W L111V, D152S, M155L,77%  8% 56% 6% 30% 5% A162W, G188A L111V, D152S, M155L, 78% 15% 64% 16% 30% 6% A162W, A216Y, K229W L111V, D152S, M155L, 66% 16% 54% 18%  25%10%  A162W, K229W L111V, D152S, M155L, 58% ND 47% ND 24% ND A162W,N189K, K229W L111V, D152S, M155L, 67%  8% 52% 4% 20% 18%  A162W, N189KL111V, D152S, M155L, 80% 10% 57% 10%  20% 11%  A162W, G188W L111V,D152S, M155L, 53% 11% 34% 7%  7% 6% A162W Wild-Type 33% 12%  3% 9% 0.6% 3%

Example 8: Purification of Aspergillus fumigatus GH61B PolypeptideVariants

Expression and purification of the wild-type Aspergillus fumigatus GH61Bpolypeptide was conducted as previously described in WO 2012/044835.

The Aspergillus fumigatus GH61B polypeptide variant strains were grownto recover culture broths for purification. Following isolation ofsingle colonies, Aspergillus oryzae PFJO218 transformants were culturedfor 4 days at 34° C. on COVE-N-GLY plates in preparation for largerscale fermentation. Spores were recovered from each plate using 0.01%TWEEN® 20. Each spore suspension (500 μl) was inoculated into 25 ml ofM400 medium in 125 ml plastic shake flasks. The transformants werefermented for 3 days at 39° C. with agitation at 150 rpm and the brothswere collected and filtered using 0.22 μm filters. The filtered culturebroths were then concentrated by centrifugal ultrafiltration usingVIVACELL® 100 5 kDa MWCO centrifugal concentration devices (SartoriusStedim, Goettingen, Germany) and then buffer exchanged into 20 mMTris-HCl pH 8.5.

The concentrated and buffer exchanged Aspergillus fumigatus GH61Bpolypeptide variants were further purified by one of two chromatographicmethods. In one method, the concentrated and buffer exchanged brothswere then each applied to a MONO Q® HR 16/10 column (GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Tris-HCl pH 8.0. Boundproteins were eluted with a linear gradient of 0-600 mM sodium chloridein 20 mM Tris-HCl pH 8.0. Fractions were analyzed by SDS-PAGE using aCRITERION® Stain-Free Tris-HCl 8-16% SDS-PAGE gel (Bio-Rad Laboratories,Inc., Hercules, Calif., USA), pooled based on the abundance of anapproximately 25 kDa band, and concentrated using VIVASPIN® 5 kDa MWCOcentrifugal concentration devices (GE Healthcare, Buckinghamshire,United Kingdom). Alternatively, the concentrated and desalted brothswere then each applied to a HILOAD® 26/60 SUPERDEX® 75 (GE Healthcare,Piscataway, N.J., USA) size exclusion column which had been equilibratedwith 20 mM Tris-HCl pH 8.0 and 150 mM NaCl. Applied proteins were elutedisocraticly using 20 mM Tris-HCl pH 8.0 and 150 mM NaCl as the mobilephase. Fractions were analyzed by SDS-PAGE using a CRITERION® Stain-FreeTris-HCl 8-16% SDS-PAGE gel, pooled based on the abundance of anapproximately 25 kDa band, and concentrated using VIVASPIN® 5 kDa MWCOcentrifugal concentration devices.

Protein concentrations were determined using a Microplate BCA™ ProteinAssay Kit in which bovine serum albumin was used as a protein standard.

Example 9: Determination of Tm (Melting Temperature) of the Aspergillusfumigatus Wild-Type GH61B Polypeptide and Aspergillus fumigatus GH61BPolypeptide Variants by Differential Scanning Calorimetry

The thermostabilities of the A. fumigatus wild-type GH61B polypeptideand the Aspergillus fumigatus GH61B polypeptide variants, which werepurified as described in Example 8, were determined by DifferentialScanning calorimetry (DSC) using a VP Differential Scanning calorimeter(MicroCal Inc., GE Healthcare, Piscataway, N.J., USA). The meltingtemperature, Tm (° C.), was taken as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heating a 1mg protein per ml solution of the enzyme in 50 mM sodium acetate pH 5.0,100 μM CuSO₄, or a 1 mg protein per ml solution of the enzyme in 50 mMsodium acetate pH 5.0, 10 mM EDTA pH 5.0, at a constant programmedheating rate. One ml of sample and reference-solutions were degassed at25° C. using a ThermoVac (MicroCal Inc., GE Healthcare, Piscataway,N.J., USA) prior to loading of sample and reference cells of thecalorimeter. Sample and reference (reference: degassed water) solutionswere manually loaded into the DSC and thermally pre-equilibrated to 25°C. before the DSC scan was performed from 25° C. to 95° C. at a scanrate of 90 K/hour. Denaturation temperatures were determined at anaccuracy of approximately +/−1° C. The results of the thermostabilitydetermination of the A. fumigatus GH61 B polypeptide variants are shownin Table 6.

TABLE 6 Melting temperatures (° C.) of the A. fumigatus GH61Bpolypeptide and variants of the A. fumigatus GH61B polypeptide, asdetermined by differential scanning calorimetry Tm + 100 μm Tm + 10 mMMutations CuSO₄ EDTA pH 5 Wild-Type 69 59 G188A 75 n.d. G188W 75 63N189K 74 63 K229W 74 63 L111V + D152S + M155L + A162W 76 66 L111V +D152S + M155L + A162W + n.d. 70 K229W L111V + D152S + M155L + A162W + 8373 G188F + K229W

Example 10: Determination of Tm (Melting Temperature) of the Aspergillusfumigatus Wild-Type GH61B Polypeptide and Aspergillus fumigatus GH61BPolypeptide Variants by Protein Thermal Unfolding Analysis

Protein thermal unfolding of the Aspergillus fumigatus GH61B polypeptidevariants was monitored using SYPRO® Orange Protein Stain (Invitrogen,Naerum, Denmark) using a StepOnePlus™ Real-Time PCR System (AppliedBiosystems Inc., Foster City, Calif., USA). In a 96-well whitePCR-plate, 15 μl of a protein sample (prepared as described in Example8) in 100 mM sodium acetate pH 5.0 was mixed (1:1) with Sypro Orange(resulting concentration=10×; stock solution=5000× in DMSO) in 20 mMEDTA. The plate was sealed with an optical PCR seal. The PCR instrumentwas set at a scan-rate of 76° C. per hour, starting at 25° C. andfinishing at 96° C. Fluorescence was monitored every 20 seconds using abuilt-in LED blue light for excitation and ROX-filter (610 nm,emission). Tm-values were calculated as the maximum value of the firstderivative (dF/dK) (Gregory et al., 2009, J. Biomol. Screen. 14: 700).The results of the thermostability determinations are shown in Table 7.

TABLE 7 Melting temperatures (° C.) of the A. fumigatus GH61Bpolypeptide and variants determined by thermal unfolding analysisMutations Tm Wild-Type 59 E105P 62 E154L 61 A216Y 60 A216L 63 K229H 61K229I 60

Example 11: Preparation of a High-Temperature Cellulase Composition

Aspergillus fumigatus GH7A cellobiohydrolase I (SEQ ID NO: 245 [genomicDNA sequence] and SEQ ID NO: 246 [deduced amino acid sequence]) wasprepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The filtered broth of the Aspergillus fumigatus GH7Acellobiohydrolase I was concentrated and buffer exchanged with 20 mMTris-HCl pH 8.0 using a tangential flow concentrator (Pall Filtron,Northborough, Mass., USA) equipped with a 10 kDa polyethersulfonemembrane (Pall Filtron, Northborough, Mass., USA).

Aspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO: 247 [genomicDNA sequence] and SEQ ID NO: 248 [deduced amino acid sequence]) wasprepared recombinantly in Aspergillus oryzae as described in WO2011/057140. The filtered broth of the Aspergillus fumigatus GH6Acellobiohydrolase II was concentrated and buffer exchanged with 20 mMTris-HCl pH 8.0 using a tangential flow concentrator equipped with a 10kDa polyethersulfone membrane.

Trichoderma reesei GH5 endoglucanase II (SEQ ID NO: 249 [genomic DNAsequence] and SEQ ID NO: 250 [deduced amino acid sequence]) was preparedrecombinantly in Aspergillus oryzae as described in WO 2011/057140.Filtered broth of the Trichoderma reesei GH5 endoglucanase II wasconcentrated and buffer exchanged with 20 mM Tris-HCl pH 8.0 using atangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane. Aspergillus fumigatus GH10 xylanase (xyn3) (SEQ ID NO: 251[genomic DNA sequence] and SEQ ID NO: 252 [deduced amino acid sequence])was prepared recombinantly according to WO 2006/078256 using Aspergillusoryzae BECh2 (WO 2000/39322) as a host. Filtered broth of theAspergillus fumigatus NN055679 GH10 xylanase (xyn3) was desalted andbuffer-exchanged with 50 mM sodium acetate pH 5.0 using a HIPREP® 26/10Desalting column (GE Healthcare, Piscataway, N.J., USA) according to themanufacturer's instructions.

Aspergillus fumigatus Cel3A beta-glucosidase was prepared as describedin Example 4.

Aspergillus fumigatus GH3 beta-xylosidase (SEQ ID NO: 253 [genomic DNAsequence] and SEQ ID NO: 254 [deduced amino acid sequence]) was preparedrecombinantly in Aspergillus oryzae as described in WO 2011/057140 andpurified according to WO 2011/057140.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard. The cellulase compositionwas composed of 44.7% Aspergillus fumigatus Cel7A cellobiohydrolase I,29.4% Aspergillus fumigatus Cel6A cellobiohydrolase II, 11.8%Trichoderma reesei GH5 endoglucanase II, 5.9% Aspergillus fumigatus GH10xylanase (xyn3), 5.9% Aspergillus fumigatus beta-glucosidase, and 2.3%Aspergillus fumigatus beta-xylosidase. The cellulase composition isdesignated herein as a “high-temperature cellulase composition”.

Example 12: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained approximately 59% cellulose, 5%hemicelluloses, and 28% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of PCS per ml of 50 mM sodiumacetate pH 5.0 buffer containing 1 mM manganese sulfate and a proteinloading of the high-temperature cellulase composition (expressed as mgprotein per gram of cellulose). Enzyme mixtures were prepared and thenadded simultaneously to all wells in a volume of 100 μl, for a finalvolume of 1 ml in each reaction. The plate was then sealed using anALPS-300™ plate heat sealer (Abgene, Epsom, United Kingdom), mixedthoroughly, and incubated at 50° C., 55° C., and 60° C. for 72 hours.All experiments were performed in triplicate.

Following hydrolysis, samples were filtered with a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered sugary aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at aflow rate of 0.6 ml per minute at 65° C., and quantitation byintegration of glucose and cellobiose signals using a refractive indexdetector (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantequivalents were used to calculate the percentage of celluloseconversion for each reaction.

All HPLC data processing was performed using MICROSOFT EXCEL™ software(Microsoft, Richland, Wash., USA). Measured sugar concentrations wereadjusted for the appropriate dilution factor. Glucose and cellobiosewere measured individually. However, to calculate total conversion theglucose and cellobiose values were combined. Cellobiose concentrationwas multiplied by 1.053 in order to convert to glucose equivalents andadded to the glucose concentration. The degree of cellulose conversionwas calculated using the following equation:

% conversion=([sample glucose concentration]/[glucose concentration in alimit digest])×100

In order to calculate % conversion, a 100% conversion point was setbased on a cellulase control of 50 mg of the cellulase composition pergram cellulose (CELLUCLAST PLUS™, Novozymes A/S, Bagsvaerd, Denmark),and all values were divided by this number and then multiplied by 100.Triplicate data points were averaged and standard deviation wascalculated.

Example 13: Effect of Addition of Aspergillus fumigatus GH61BPolypeptide Variants in the Conversion of PCS by a High-TemperatureCellulase Composition at 50° C., 55° C., and 60° C.

A. fumigatus GH61B wild-type polypeptide and Aspergillus fumigatus GH61Bpolypeptide variants N189K, G188W, K229W, and G188A were evaluated fortheir ability to enhance the hydrolysis of PCS by the high temperaturecellulase composition (Example 11) at 50° C., 55° C., or 60° C. Thepretreated corn stover hydrolysis assay was performed as described inExample 12. The high temperature composition when loaded at 3 mg totalprotein per gram cellulose in the assay had the following enzymeloadings per gram cellulose: 1.34 mg of A. fumigatus cellobiohydrolase Iper gram cellulose, 0.88 mg of A. fumigatus cellobiohydrolase II pergram cellulose, 0.18 mg of A. fumigatus beta-glucosidase per gramcellulose, 0.18 mg of Aspergillus fumigatus GH10 xylanase (Xyl3) pergram cellulose, 0.18 mg of A. fumigatus beta-xylosidase per gramcellulose, and 0.35 mg of T. reesei CELSA endoglucanase II per gramcellulose.

The conversion of pretreated corn stover by the high temperaturecellulase composition (3 mg protein per gram cellulose); the combinationof the high temperature cellulase composition (3 mg protein per gramcellulose) and A. fumigatus GH61B wild-type polypeptide (0.25 mg proteinper gram cellulose); the combination of the high temperature cellulasecomposition (3 mg protein per gram cellulose) and Aspergillus fumigatusGH61B variant N168K polypeptide (0.25 mg protein per gram cellulose);the combination of the high temperature cellulase composition (3 mgprotein per gram cellulose) and Aspergillus fumigatus GH61B variantG167W polypeptide (0.25 mg protein per gram cellulose); the combinationof the high temperature cellulase composition (3 mg protein per gramcellulose) and Aspergillus fumigatus GH61B variant K208W polypeptide(0.25 mg protein per gram cellulose); and the combination of the hightemperature cellulase composition (3 mg protein per gram cellulose) andAspergillus fumigatus GH61B variant G167A polypeptide (0.25 mg proteinper gram cellulose) were assayed as described in Example 12. Data werecollected and analyzed, as described in Example 12, after 72 hours ofincubation at 50° C., 55° C., or 60° C. These results are shown in FIG.6.

The high temperature cellulase composition (3 mg protein per gramcellulose) resulted in conversions of 46.8±0.3%, 47.9±1.0%, and45.2±0.2% at 50° C., 55° C., or 60° C., respectively, of the pretreatedcorn stover.

The combination of the high temperature cellulase composition (3 mgprotein per gram cellulose) and A. fumigatus GH61B wild-type polypeptide(0.25 mg protein per gram cellulose) resulted in conversions of55.3±0.3%, 56.2±0.7%, and 51.3±0.4% at 50° C., 55° C., or 60° C.,respectively, of the pretreated corn stover.

The combination of the high temperature cellulase composition (3 mgprotein per gram cellulose) and A. fumigatus GH61B variant N189Kpolypeptide (0.25 mg protein per gram cellulose) resulted in conversionsof the pretreated corn stover of 56.3±0.5%, 57.5±0.3%, and 51.3±0.3% at50° C., 55° C., or 60° C., respectively, of the pretreated corn stover.

The combination of the high temperature cellulase composition (3 mgprotein per gram cellulose) and A. fumigatus GH61B variant G188Wpolypeptide (0.25 mg protein per gram cellulose) resulted in conversionsof 55.8±0.4%, 57.5±0.4%, and 52.4±0.04% at 50° C., 55° C., or 60° C.,respectively, of the pretreated corn stover.

The combination of the high temperature cellulase composition (3 mgprotein per gram cellulose) and A. fumigatus GH61B variant K229Wpolypeptide (0.25 mg protein per gram cellulose) resulted in conversionsof 56.8±0.7%, 57.6±0.1%, and 53.0±0.3% at 50° C., 55° C., or 60° C.,respectively, of the pretreated corn stover.

The combination of the high temperature cellulase composition (3 mgprotein per gram cellulose) and A. fumigatus GH61B variant G188Apolypeptide (0.25 mg protein per gram cellulose) resulted in conversionsof 55.9±0.3%, 56.9±0.5%, and 51.3±0.3% at 50° C., 55° C., or 60° C.,respectively, of the pretreated corn stover.

Example 14: Construction of Penicillium emersonii GH61A and Thermoascusaurantiacus GH61A Polypeptide Variants

Variants of the Penicillium emersonii GH61A polypeptide (SEQ ID NO: 35[DNA sequence] and SEQ ID NO: 36 [amino acid sequence]), and Thermoascusaurantiacus GH61A polypeptide (SEQ ID NO: 13 [DNA sequence] and SEQ IDNO: 14 [amino acid sequence]) were constructed by performingsite-directed mutagenesis on plasmids pMMar45 and pDFng113,respectively, using a QUIKCHANGE® Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif., USA) as described in Example 7. A summaryof the primers used for the site-directed mutagenesis and the variantsobtained are shown in Table 8. The same protocol described in Example 7was used.

The resulting mutant plasmid DNAs were prepared using a BIOROBOT® 9600.Each mutant plasmid was sequenced using a 3130xl Genetic Analyzer toverify the substitutions. The sequencing primers 996271 and pALLO2 3′were used for verification.

TABLE 8 Variant Variant Template Amino Acid Primer Plasmid BackboneSubstitution ID Primer Sequence Name Penicillium D109P 615190GCAGTGGACGCCGTGGCCGCCGAG pLSBF07-1 emersonii CCACCACGGACCCGTCAT (SEQ IDGH61A NO: 255) 615201 ATGACGGGTCCGTGGTGGCTCGGCGGCCACGGCGTCCACTGC (SEQ ID NO: 256) Penicillium D109K 615191GCAGTGGACGCCGTGGCCGAAGAG pLSBF07-2 emersonii CCACCACGGACCCGTCAT (SEQ IDGH61A NO: 257) 615202 ATGACGGGTCCGTGGTGGCTCTTCGGCCACGGCGTCCACTGC (SEQ ID NO: 258) Penicillium N192A 615193CATCGCCCTGCACTCGGCCGCCAAC pLSBF07-4 emersonii AAGGACGGCGCCCAGAAC (SEQ IDGH61A NO: 259) 615204 GTTCTGGGCGCCGTCCTTGTTGGCGGCCGAGTGCAGGGCGATG (SEQ ID NO: 260) Penicillium N192W 615194CATCGCCCTGCACTCGGCCTGGAAC pLSBF07-5 emersonii AAGGACGGCGCCCAGAAC (SEQ IDGH61A NO: 261) 615205 GTTCTGGGCGCCGTCCTTGTTCCAGGCCGAGTGCAGGGCGATG (SEQ ID NO: 262) Penicillium N193K 615195CGCCCTGCACTCGGCCAACAAGAAG pLSBF07-6 emersonii GACGGCGCCCAGAACTAC (SEQ IDGH61A NO: 263) 615206 GTAGTTCTGGGCGCCGTCCTTCTTGTTGGCCGAGTGCAGGGCG (SEQ ID NO: 264) Thermoascus D105K 615253GCTTCAATGGACTCCATGGCCTAAA pDFng113-1 aurantiacus TCTCACCATGGCCCAGTTATCAGH61A (SEQ ID NO: 265) 615254 TGATAACTGGGCCATGGTGAGATTTAGGCCATGGAGTCCATTGAAGC (SEQ ID NO: 266) Thermoascus D105P 615255GCTTCAATGGACTCCATGGCCTCCT pDFng113-3 aurantiacus TCTCACCATGGCCCAGTTATCAGH61A (SEQ ID NO: 267) 615256 TGATAACTGGGCCATGGTGAGAAGGAGGCCATGGAGTCCATTGAAGC (SEQ ID NO: 268) Thermoascus Q188W 615273GAGATTATTGCTCTTCACTCAGCTTG pDFng113- aurantiacus GAACCAGGATGGTGCCCAGAAC28 GH61A (SEQ ID NO: 269) 615275 GTTCTGGGCACCATCCTGGTTCCAAGCTGAGTGAAGAGCAATAATCTC (SEQ ID NO: 270)

The P. emersonii GH61A polypeptide variants and T. aurantiacus GH61Apolypeptide variants were expressed using Aspergillus oryzae PFJO218 ashost was performed according to the procedure described in Example 3.

Example 15: Preparation of Penicillium emersonii Wild-Type GH61APolypeptide and P. emersonii GH61A Polypeptide Variants

The P. emersonii GH61A polypeptide wild-type and variant strains weregrown as described in Example 8 to recover culture broths forpurification.

The filtered culture broths were then concentrated by centrifugalultrafiltration using VIVACELL® 20 5 kDa MWCO centrifugal concentrationdevices (Sartorius Stedim, Goettingen, Germany) and then bufferexchanged into 20 mM Tris-HCl pH 8.0. Protein concentrations weredetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

In the cases of P. emersonii GH61A wild-type polypeptide, P. emersoniiGH61A polypeptide variant N192W, and P. emersonii GH61A polypeptidevariant N193K, further purification was conducted by application of theconcentrated and buffer exchanged broths to HITRAP® Q SEPHAROSE® FastFlow columns (GE Healthcare, Piscataway, N.J., USA) equilibrated with 20mM Tris-HCl pH 8.0. Bound proteins were eluted with a linear gradient of0-500 mM sodium chloride in 20 mM Tris-HCl pH 8.0. Fractions wereanalyzed by SDS-PAGE using a CRITERION® Tris-HCl 8-16% SDS-PAGE gel(Bio-Rad Laboratories, Inc., Hercules, Calif., USA), pooled based on theabundance of an approximately 25 kDa band, and concentrated usingVIVASPIN® 5 kDa MWCO centrifugal concentration devices. Proteinconcentrations were determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 16: Preparation of Thermoascus aurantiacus GH61A PolypeptideVariants

The T. aurantiacus GH61A polypeptide wild-type and variant strains weregrown as described in Example 8 to recover culture broths forpurification.

The filtered culture broths were then concentrated by centrifugalultrafiltration using VIVACELL® 20 5 kDa MWCO centrifugal concentrationdevices (Sartorius Stedim, Goettingen, Germany) and then bufferexchanged into 20 mM Tris-HCl pH 8.0. Protein concentrations weredetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

The purification was conducted by application of the concentrated andbuffer exchanged broths to HITRAP® Q SEPHAROSE® Fast Flow columnsequilibrated with 20 mM Tris-HCl pH 8.0. Bound proteins were eluted witha linear gradient of 0-500 mM sodium chloride in 20 mM Tris-HCl pH 8.0.Fractions were analyzed by SDS-PAGE using a CRITERION® Tris-HCl 8-16%SDS-PAGE gel, pooled based on the abundance of an approximately 25 kDaband, and concentrated using VIVASPIN® 5 kDa MWCO centrifugalconcentration devices. Protein concentrations were determined using aMicroplate BCA™ Protein Assay Kit in which bovine serum albumin was usedas a protein standard.

Example 17: Determination of Tm (Melting Temperature) of the P.emersonii Wild-Type GH61A Polypeptide and P. emersonii GH61A PolypeptideVariants by Differential Scanning Calorimetry

The thermostabilities of the P. emersonii wild-type GH61A polypeptideand the P. emersonii GH61A polypeptide variants, purified as describedin Example 15, were determined by Differential Scanning calorimetry(DSC) using a VP Differential Scanning calorimeter. The meltingtemperature, Tm (° C.), was taken as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heating a 1mg protein per ml solution of the enzyme in 50 mM sodium acetate pH 5.0,100 μM CuSO₄, or a 1 mg protein per ml solution of the enzyme in 50 mMsodium acetate pH 5.0, 10 mM EDTA pH 5.0, at a constant programmedheating rate. One ml of sample and reference-solutions were degassed at25° C. using a ThermoVac prior to loading of sample and reference cellsof the calorimeter. Sample and reference (reference: degassed water)solutions were manually loaded into the DSC and thermallypre-equilibrated to 25° C. before the DSC scan was performed from 25° C.to 95° C. at a scan rate of 90 K/hour. Denaturation temperatures weredetermined at an accuracy of approximately +/−1° C.

The results of the thermostability determination of the P. emersoniiGH61A polypeptide variants are shown in Table 9.

TABLE 9 Melting temperatures (° C.) of P. emersonii GH61A polypeptideand variants of P. emersonii GH61A polypeptide, as determined bydifferential scanning calorimetry Enzyme sample Tm + 100 μM CuSO₄ P.emersonii GH61A 82 P. emersonii GH61A N192W 84 P. emersonii GH61A N193K83

Example 18: Determination of Tm (Melting Temperature) of Penicilliumemersonii GH61A and Thermoascus aurantiacus GH61A Polypeptide Variantsby Protein Thermal Unfolding Analysis

Protein thermal unfolding of the Penicillium emersonii GH61A andThermoascus aurantiacus GH61A polypeptide variants was monitored usingSYPRO® Orange Protein Stain and was performed using a StepOnePlus™Real-Time PCR System as described as Example 10. P. emersonii GH61Awild-type polypeptide and P. emersonii GH61A polypeptide variants wereconcentrated and buffer exchanged as described in Example 15. T.aurantiacus GH61A wild-type polypeptide and T. aurantiacus GH61Apolypeptides variants were purified as described in Example 16. Theresults of the thermostability determination are shown in Table 10.

TABLE 10 Melting temperature (° C.) of Penicillium emersonii GH61A andThermoascus aurantiacus GH61A polypeptide variants by protein thermalunfolding analysis Protein backbone Sample type Mutations Tm P.emersonii GH61A Concentrated Wild-Type 69 P. emersonii GH61AConcentrated D109P 71 P. emersonii GH61A Concentrated D109K 70 P.emersonii GH61A Concentrated N192A 70 P. emersonii GH61A ConcentratedN192W 71 P. emersonii GH61A Concentrated N193K 71 T. aurantiacus GH61APurified WT 72 T. aurantiacus GH61A Purified D105K 74 T. aurantiacusGH61A Purified D105P 74 T. aurantiacus GH61A Purified Q188W 74

Example 19: Construction of Expression Vectors pDFng153-4, pDFng154-17,and pDFng155-33

Plasmids pDFng153-4 (FIG. 7), pDFng154-17 (FIG. 8), and pDFng155-33(FIG. 9) were constructed as described below for expression of theThermoascus aurantiacus GH61A polypeptide, Penicillium emersonii GH61Apolypeptide, and Aspergillus aculeatus GH61 polypeptide, respectively,and generation of the variants listed in Table 11. The plasmids wereconstructed using plasmid pBGMH16 (FIG. 10).

Plasmid pBGMH16 was constructed according to the protocol describedbelow. A Nb.Btsl recognition site in pUC19 was removed by PCR amplifyingpUC19 with primer pair BGMH24/BGMH25 followed by the uracil-specificexcision reagent USER™ based cloning (New England BioLabs, Ipswich,Mass., USA). Plasmid pUC19 is described in Yanisch-Perron et al., 1985,Gene 33(1):103-19.

BGMH 24 ATGCAGCGCUGCCATAACCATGAGTGA (SEQ ID NO: 271) BGMH 25AGCGCTGCAUAATTCTCTTACTGTCATG (SEQ ID NO: 272)Underlined sequence is used in the USER™ assisted fusion of the PCRfragments creating pBGMH13. USER™ (Uracil-Specific Excision Reagent)Enzyme (New England Biolabs, Ipswich, Mass., USA) generates a singlenucleotide gap at the location of a uracil. USER™ Enzyme is a mixture ofUracil DNA glycosylase (UDG) and the DNA glycosylase-lyase EndonucleaseVIII. UDG catalyzes the excision of a uracil base, forming an abasic(apyrimidinic) site while leaving the phosphodiester backbone intact.The lyase activity of Endonuclease VIII breaks the phosphodiesterbackbone at the 3′ and 5′ sides of the basic site so that base-freedeoxyribose is released.

The amplification reaction was composed of 100 ng of each primer, 10 ngof pUC19, 1× PfuTurbo® C_(x) Reaction Buffer (Stratagene, La Jolla,Calif., USA), 2.5 μl of a blend of dATP, dTTP, dGTP, and dCTP, each at10 mM, and 2.5 units of PfuTurbo® C_(x) Hot Start DNA Polymerase(Stratagene, La Jolla, Calif., USA), in a final volume of 50 μl. Thereaction was performed using a EPPENDORF® MASTERCYCLER® 5333 programmedfor 1 cycle at 95° C. for 2 minutes; 32 cycles each at 95° C. for 30seconds, 55° C. for 30 seconds, and 72° C. for 3 minutes; and a finalelongation at 72° C. for 7 minutes. Five μl of 10× NEBuffer 4 (NewEngland Biolabs, Inc., Ipswich, Mass., USA), and 20 units of Dpn I wereadded and incubated 1 hour at 37° C. The Dpn I was inactivated at 80° C.for 20 minutes. A total of 100 ng of the PCR product and 1 unit of USER™enzyme in a total volume of 10 μl were incubated 20 minutes at 37° C.followed by 20 minutes at 25° C. Ten μl were transformed into ONE SHOT®TOP10 competent cells. This resulted in plasmid pBHMG13.

Plasmid pBGMH14 contains part of pBGMH13 as vector backbone and a PacI/Nt.BbvCI USER™ cassette (Hansen et al., 2011, Appl. Environ.Microbiol. 77(9): 3044-51) which is flanked by part of the A. oryzaeniaD gene on one side and part of the A. oryzae niiA gene on the otherside. The Pac I/Nt.BbvCI USER™ cassette can be linearized with Pac I andNt.BbvCI and a PCR product with compatible overhangs can be cloned intothis site (New England Biolabs, Ipswich, Mass., USA).

BGMH 27 AATTAAGU CCTCAGCGTGATTTAAAACGCCATTGCT (SEQ ID NO: 273) BGMH 28ACTTAATU AAACCCTCAGCGCAGTTAGGTTGGTGTTCTTCT (SEQ ID NO: 274) BGMH 29AGCTCAAGGAUACCTACAGTTATTCGAAA (SEQ ID NO: 275) BGMH 30ATCCTTGAGCUGTTTCCTGTGTGAAATTGTTATCC  (SEQ ID NO: 276) BGMH 31ATCTCCTCUGCTGGTCTGGTTAAGCCAGCCCCGACAC (SEQ ID NO: 277) BGMH 32AGAGGAGAUAATACTCTGCGCTCCGCC (SEQ ID NO: 278)

Underlined sequence was used in the USER™ assisted fusion of the threefragments. The sequence marked in bold was used to introduce aPacI/Nt.BbvCI USER™ cassette (Hansen et al., 2011, supra) between theniiA and niaD fragments.

An Aspergillus oryzae niiA fragment was generated using primers BGMH27and BGMH29. The primer pair BGMH28/BGMH32 was used to amplify theAspergillus oryzae niaD gene region and primer-pair BGMH30/BGMH31 wasused to amplify the plasmid backbone region.

Genomic DNA from A. oryzae BECH2 (WO 00/39322) was purified using aFASTDNA™ 2 ml SPIN Kit for Soil (MP Biomedicals, Santa Ana, Calif.,USA).

The amplification reaction was composed of 100 ng of each primer,template DNA (pBGMH13 or A. oryzae BECH2 genomic DNA), 1× PfuTurbo® C,Reaction Buffer, 2.5 μl of a blend of dATP, dTTP, dGTP, and dCTP, eachat 10 mM, and 2.5 units of PfuTurbo® C, Hot Start DNA Polymerase, in afinal volume of 50 μl. The reaction was performed using a EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 2 minutes; 32cycles each at 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 4 minutes; and a final elongation at 72° C. for 10 minutes. For PCRtubes where template DNA was a plasmid, 5 μl of 10× NEBuffer 4 and 20units of Dpn I were added and incubated 1 hour at 37° C. The Dpn I wasinactivated at 80° C. for 20 minutes. Fifty ng of each of the PCRproducts and 1 unit of USER™ enzyme in a total volume of 10 μl wereincubated for 20 minutes at 37° C. followed by 20 minutes at 25° C. Then10 μl were transformed into ONE SHOT® TOP10 competent cells. The threefragments were fused by uracil-specific excision reagent based cloningresulting in pBGMH14.

The promoter P13amy is a derivative of the NA-2 tpi promoter frompJaL676 (WO 2003/008575). The A. niger AMG terminator used is describedby Christensen et al., 1988, Nature Biotechnology 6: 141-1422.

The P13amy promoter and AMG terminator were cloned into thePacI/Nt.BbvCI USER™ cassette in pBGMH14. The primers were designed sothat an AsiSI/Nb.Btsl USER™ cassette (Hansen et al., 2011, supra) wasintroduced between the promoter and terminator.

BGMH 49 GGGTTTAAUCCTCACACAGGAAACAGCTATGA (SEQ ID NO: 279) BGMH 50AGTGTCTGCGAU CGCTCTCACTGCCCCCAGTTGTGTATATAG AGGA (SEQ ID NO: 280)BGMH 51 ATCGCAGACACU GCTGGCGGTAGACAATCAATCCAT (SEQ ID NO: 281) BGMH 52GGACTTAAUGGATCTAAGATGAGCTCATGGCT (SEQ ID NO: 282)

Underlined sequence was used in the USER™ assisted fusion of the twofragments into a PacI/Nt.BbvCI digested pBGMH14. The sequence marked inbold was used to introduce a AsiSI/Nb.Btsl USER™ cassette (Hansen etal., 2011, supra) between the promoter and terminator.

Promoter P13amy and AMG terminator was PCR amplified using the primerpair BGMH49/BGMH50 to amplify promoter P13amy and the primer pairBGMH51/BGMH52 to amplify the AMG terminator. The amplification reactionwas composed of 100 ng of each primer, template DNA, 1× PfuTurbo® C_(x)Reaction Buffer, 2.5 μl of a blend of dATP, dTTP, dGTP, and dCTP, eachat 10 mM, and 2.5 units of PfuTurbo® C_(x) Hot Start DNA Polymerase, ina final volume of 50 μl. The reaction was performed using a EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 2 minutes; 32cycles each at 95° C. for 30 seconds, 55° C. for 30 seconds, and 72° C.for 45 seconds; and a final elongation at 72° C. for 3 minutes. Then 5μl of 10× NEBuffer 4 and 20 units of Dpn I were added and incubated 1hour at 37° C. The Dpn I was inactivated at 80° C. for 20 minutes.

The two fragments were fused into PacI/Nt.BbvCI digested pBGMH14 byUSER™ based cloning method in a reaction composed of 10 ng ofPacI/Nt.BbvCI digested pBGMH14, 50 ng of each of the two PCR products,and 1 unit of USER™ enzyme in a total volume of 10 μl. The reaction wasincubated for 20 minutes at 37° C. followed by 20 minutes at 25° C. Then10 μl were transformed into ONE SHOT® TOP10 competent cells. E. colitransformants were selected on 2XYT+Amp agar plates and plasmid DNA wasprepared using QIAPREP® Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA). Plasmid pBGMH16 was confirmed by sequencing analysis.

DNA sequencing was performed using a Model 377 XL Automated DNASequencer and dye-terminator chemistry (Giesecke et al., 1992, supra).Sequencing primers used for verification of niiA, niaD, the P13amypromoter, AsiSI/Nb.Btsl USER™ cassette, and AMG terminator sequence inBGMH16 are shown below.

BGMH 36 ACGCCATTGCTATGATGCTTGAAG (SEQ ID NO: 283) BGMH 37TGGTGAGGTGCTATCGTCCTT (SEQ ID NO: 284) BGMH 38 CTTCCTGTAGGTGCACCGAAG(SEQ ID NO: 285) BGMH 39 ACAGAACGATATCGGACCTCG (SEQ ID NO: 286) BGMH 40TCGTTATGTTAAGTCTTCTATCA (SEQ ID NO: 287) BGMH 41 AGAGCTCGAAGTTCCTCCGAG(SEQ ID NO: 288) BGMH 42 TATCACGAGGCCCTTTCGTCTC (SEQ ID NO: 289) BGMH 43TCCGTCGGCTCCTCTCCTTCGT (SEQ ID NO: 290) BGMH 44 TGCATATCCTCTGACAGTATATGA(SEQ ID NO: 291) BGMH 45 CAGTGAAGAGGGCAGTCGATAGT (SEQ ID NO: 292)BGMH 46 ACGAGGAACATGGCTATCTGGA (SEQ ID NO: 293) BGMH 47TCAGCTCATTCTGGGAGGTGGGA (SEQ ID NO: 294) BGMH 48 ACTCCAGGATCCTTTAAATCCA(SEQ ID NO: 295) BGMH 53 ACTGGCAAGGGATGCCATGCT (SEQ ID NO: 296) BGMH 54TGATCATATAACCAATTGCCCT (SEQ ID NO: 297) BGMH 55 AGTTGTGTATATAGAGGATTGA(SEQ ID NO: 298) BGMH 56 TGGTCCTTCGCTCGTGATGTGGA (SEQ ID NO: 299)BGMH 57 AGTCCTCAGCGTTACCGGCA (SEQ ID NO: 300) BGMH 58ACCCTCAGCTGTGTCCGGGA (SEQ ID NO: 301) BGMH 59 TGGTATGTGAACGCCAGTCTG(SEQ ID NO: 302)

Plasmid pBGMH16 contains flanking regions designed to repair the niiAgene and niaD gene in Aspergillus oryzae COLs1300. Plasmid pBGMH16 wasdigested with Asi Si and Nb. Bts I to linearize the plasmid and createsingle stranded overhangs so that a PCR product with compatibleoverhangs can be cloned into this site by USER™ cloning (New EnglandBiolabs, Inc., Ipswich, Mass., USA). The digested plasmid was purifiedusing a DNA Purification Kit (QIAGEN Inc., Valencia, Calif., USA)according to the manufacturer's instructions.

The T. aurantiacus GH61A polypeptide coding sequence (SEQ ID NO: 13[genomic DNA sequence] and SEQ ID NO: 14 [deduced amino acid sequence]),P. emersonii GH61A polypeptide coding sequence (SEQ ID NO: 35 [genomicDNA sequence] and SEQ ID NO: 36 [deduced amino acid sequence]), and A.aculeatus GH61 polypeptide coding sequence (SEQ ID NO: 67 [genomic DNAsequence] and SEQ ID NO: 68 [deduced amino acid sequence]) wereamplified from source plasmids described below using the primers shownin Table 11. Bold letters represent coding sequence. The singledeoxyuridine (U) residue inserted into each primer is the U that isexcised from the PCR products using the USER™ enzyme (New EnglandBiolabs, Inc., Ipswich, Mass., USA) to obtain overhangs for theinsertion site. The underline letters represent a His tag. The remainingsequences are homologous to insertion sites of pBGMH16 for expression ofthe GH61 polypeptides.

TABLE 11 GH61 Source origin Template Plasmid Primer ID Primer SequenceThermoascus pDFng113 pDFng153-4 TaGH61_USE AGAGCGA(U)ATGTCCTTTTCCaurantiacus Example 1 RtagF AAGATAAT (SEQ ID NO: 303) GH61A TaGH61_USETCTGCGA(U)TTA GTGATGGTG R_HIStagR GTGATGATG ACCAGTATACAGAGGAGGAC (SEQ ID NO: 304) Penicillium pMMar45 pDFng154-17 PeGH61_USEAGAGCGA(U)ATGCTGTCTTCG emersonii Example 1 RtagFACGACTCG (SEQ ID NO: 305) GH61A PeGH61_USE TCTGCGA(U)CTA GTGATGGTGR_HIStagR GTGATGATG GAACGTCGGCT CAGGCGGCC (SEQ ID NO: 306) AspergillusXyz1566 pDFng155-33 AaGH61_USE AGAGCGA(U)ATGTCTGTTGCT aculeatus(WO 2012/ RtagF AAGTTTGCTGGTG (SEQ ID GH61 030799) NO: 307) AaGH61_USETCTGCGA(U)TTA GTGATGGTG R_HIStagR GTGATGATG GGCGGAGAGGTCACGGGCGT (SEQ ID NO: 308)

Construction of plasmid pDFng153-4 containing the Thermoascusaurantiacus GH61A polypeptide coding sequence is described below. The T.aurantiacus GH61A polypeptide coding sequence was amplified from plasmidpDFng113 using the primers shown in Table 11 with overhangs designed forcloning into plasmid pBGMH16. The amplification was composed of 100 ngof each primer listed in Table 11, 30 ng of pDFng113, 1× PfuTurbo® C_(x)Reaction Buffer, 2.5 μl of a blend of dATP, dTTP, dGTP, and dCTP, eachat 10 mM, and 2.5 units of PfuTurbo® C, Hot Start DNA Polymerase, in afinal volume of 50 μl. The amplification was performed using anEPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 2minutes; 30 cycles each at 95° C. for 30 seconds, 57.7° C. for 30seconds, and 72° C. for 1.5 minutes; and a final elongation at 72° C.for 10 minutes. The heat block then went to a 10° C. soak cycle.

The PCR reaction was analyzed by 0.7% agarose gel electrophoresis usingTBE buffer where an approximately 894 bp PCR product band was observed.The PCR reaction was then digested with 1 μl of Dpn I and 4.5 μl ofNEBuffer 4 at 37° C. overnight and purified using a QIAGEN® PurificationKit according to the manufacturer's instructions.

The homologous ends of the 894 bp PCR reaction and the AsiSI and Nb.Btsldigested pBGMH16 were joined together in a reaction composed of 10 μl ofthe PCR containing the 894 bp PCR product, 1 μl of the AsiSI and Nb.Btsldigested plasmid pBGMH16, and 1 μl of USER™ enzyme (New England Biolabs,Inc., Ipswich, Mass., USA). The reaction was incubated for 15 minutes at37° C., followed by 15 minutes at 25° C. Ten μl of the reaction weretransformed into E. coli XL10-GOLD® Super Competent Cells according tothe manufacturer's instructions. E. coli transformants were selected on2XYT+Amp agar plates. Plasmid DNA from several of the resulting E. colitransformants was prepared using a BIOROBOT® 9600. The T. aurantiacusGH61A polypeptide coding sequence insert was confirmed by DNA sequencingusing a Model 377 XL Automated DNA Sequencer and dye-terminatorchemistry (Giesecke et al., 1992, supra). The sequencing primers shownbelow were used for verification of the gene insert and sequence.

Primer TaGH61seqF: (SEQ ID NO: 309) CCCAGTTATCAACTACCTTGPrimer pBGMH16seqF: (SEQ ID NO: 310) CTCAATTTACCTCTATCCACPrimer pBGMH16seqR: (SEQ ID NO: 311) TATAACCAATTGCCCTCATC

A plasmid containing the correct T. aurantiacus GH61A polypeptide codingsequence was selected and designated pDFng153-4.

Construction of plasmid pDFng154-17 containing the Penicillium emersoniiGH61A polypeptide coding sequence is described below. The P. emersoniiGH61A polypeptide coding sequence was amplified from plasmid pMMar45using the primers shown in Table 11 with overhangs designed for cloninginto plasmid pBGMH16. The amplification was composed of 100 ng of eachprimer listed in Table 11, 30 ng of pMMar45, 1× PfuTurbo® C_(x) ReactionBuffer, 2.5 μl of a blend of dATP, dTTP, dGTP, and dCTP, each at 10 mM,and 2.5 units of PfuTurbo® C_(x) Hot Start DNA Polymerase, in a finalvolume of 50 μl. The amplification was performed using an EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 95° C. for 2 minutes; 30cycles each at 95° C. for 30 seconds, 64.1° C. for 30 seconds, and 72°C. for 1.5 minutes; and a final elongation at 72° C. for 10 minutes. Theheat block then went to a 10° C. soak cycle.

The PCR reaction was analyzed by 0.7% agarose gel electrophoresis usingTBE buffer where an approximately 930 bp PCR product band was observed.The PCR reaction was then digested with 1 μl of Dpn I and 4.5 μl ofNEBuffer 4 at 37° C. overnight and purified using a QIAGEN® PurificationKit according to the manufacturer's instructions.

The homologous ends of the 930 bp PCR reaction and the AsiSI and Nb.Btsldigested pBGMH16 were joined together in a reaction composed of 10 μl ofthe PCR containing the 930 bp PCR product, 1 μl of the AsiSI and Nb.Btsldigested pBGMH16, and 1 μl of USER™ enzyme. The reaction was incubatedfor 15 minutes at 37° C., followed by 15 minutes at 25° C. Ten μl of thereaction were transformed into E. coli XL10-GOLD® Super Competent Cellsaccording to the manufacturer's instructions. E. coli transformants wereselected on 2XYT+Amp agar plates. Plasmid DNA from several of theresulting E. coli transformants was prepared using a BIOROBOT® 9600. TheP. emersonii GH61A polypeptide coding sequence insert was confirmed byDNA sequencing using a Model 377 XL Automated DNA Sequencer anddye-terminator chemistry (Giesecke et al., 1992, supra). The sequencingprimers pBGMH16seqF and pBGMH16seqR and primer PeGH61seqF shown belowwere used for verification of the gene insert and sequence.

PeGH61seqF: (SEQ ID NO: 312) GCACCGTCGAGCTGCAGTGG

A plasmid containing the correct P. emersonii GH61A polypeptide codingsequence was selected and designated pDFng154-17.

Construction of plasmid pDFng155-33 containing the Aspergillus aculeatusGH61A polypeptide coding sequence is described below. The A. aculeatusGH61A polypeptide coding sequence was amplified from plasmid Xyz1566 (WO2012/030799 Example 3, P23NJ4 gene) using primers shown in Table 11 withoverhangs designed for cloning into plasmid pBGMH16. The amplificationreaction was composed of 100 ng of each primer listed in Table 11, 30 ngof plasmid Xyz1566, 1× PfuTurbo® C, Reaction Buffer, 2.5 μl of a blendof dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 2.5 units of PfuTurbo®C, Hot Start DNA Polymerase, in a final volume of 50 μl. Theamplification was performed using an EPPENDORF® MASTERCYCLER® 5333programmed for 1 cycle at 95° C. for 2 minutes; 30 cycles each at 95° C.for 30 seconds, 63.4° C. for 30 seconds, and 72° C. for 1.5 minutes; anda final elongation at 72° C. for 10 minutes. The heat block then went toa 10° C. soak cycle.

The PCR reaction was analyzed by 0.7% agarose gel electrophoresis usingTBE buffer where an approximately 1.3 kb PCR product band was observed.The PCR reaction was then digested with 1 μl of Dpn I and 4.5 μl ofNEBuffer 4 at 37° C. overnight and purified using a QIAGEN® PurificationKit according to the manufacturer's instructions.

The homologous ends of the 1.3 kb PCR reaction and the digested pBGMH16were joined together in a reaction composed of 10 μl of the PCRcontaining the 1.3 kb PCR product, 1 μl of the digested pBGMH16, and 1μl of USER™ enzyme. The reaction was incubated for 15 minutes at 37° C.,followed by 15 minutes at 25° C. Ten μl of the reaction were transformedinto E. coli XL10-GOLD® Super Competent Cells according to themanufacturer's instructions. E. coli transformants were selected on2XYT+Amp agar plates. Plasmid DNA from several of the resulting E. colitransformants was prepared using a BIOROBOT® 9600. The A. aculeatusGH61A polypeptide coding sequence insert was confirmed by DNA sequencingusing a Model 377 XL Automated DNA Sequencer and dye-terminatorchemistry (Giesecke et al., 1992, supra). The sequencing primerspBGMH16seqF and pBGMH16seqR and primer AaGH61seqF shown below were usedfor verification of the gene insert and sequence.

Primer AaGH61seqF: (SEQ ID NO: 313) CCTTGCCAACTGCAATGGTG

A plasmid containing the correct A. aculeatus GH61A polypeptide codingsequence was selected and designated pDFng155-33.

Example 20: Construction of the Thermoascus aurantiacus GH61A andPenicillium emersonii GH61A Polypeptide Variants

Variants of the T. aurantiacus GH61A and P. emersonii GH61A polypeptideswere constructed by performing site-directed mutagenesis on plasmidspDFng153-4 and pDFng154-17, respectively, according to the proceduredescribed in Example 7 using the primers described in Table 12.

The sequencing primers pBGMH16seqF, pBGMH16seqR, and TaGH61seqF (usedonly for T. aurantiacus GH61A variants), and primer PeGH61seqR shownbelow (used only for P. emersonii GH61A variants) were used forverification.

PeGH61seqR (only used for P. emersonii GH61A variants): (SEQ ID NO: 314)GCACCGTCGAGCTGCAGTGG

TABLE 12 Variant Amino Variant Template Acid Primer Plasmid BackboneSubstitution ID Primer Sequence Name Thermoascus Q188F 1202295ATTATTGCTCTTCACTCAGCTTTCAACCAGGA TaSDM2 aurantiacusTGGTGCCCAGAAC (SEQ ID NO: 315) GH61A 1202296GTTCTGGGCACCATCCTGGTTGAAAGCTGAG (pDFng153-4)TGAAGAGCAATAAT (SEQ ID NO: 316) Q188M 1202297ATTATTGCTCTTCACTCAGCTATGAACCAGGA TaSDM3 TGGTGCCCAGAAC (SEQ ID NO: 317)1202298  GTTCTGGGCACCATCCTGGTTCATAGCTGAG TGAAGAGCAATAAT (SEQ ID NO: 318)Penicillium N192M 1202305 CCCTGCACTCGGCCATGAACAAGGACGGCG PeSDM6emersonii C (SEQ ID NO: 319) GH61A 1202306GCGCCGTCCTTGTTCATGGCCGAGTGCAGG (pDFng154- G (SEQ ID NO: 320) 17) N193H1202307 GCACTCGGCCAACCACAAGGACGGCGCCC PeSDM7 (SEQ ID NO: 321) 1202308GGGCGCCGTCCTTGTGGTTGGCCGAGTGC (SEQ ID NO: 322)

PCR fragments were amplified from the mutant plasmids, the T.aurantiacus GH61A polypeptide plasmid pDFng153-4, and the P. emersoniiGH61A polypeptide plasmid pDFng154-17 for A. oryzae COLs1300transformation. The amplification was composed of 10 μM each of primers1201513 and 1201514 (see below), 10 ng of either pDFng153-4,pDFng154-17, or one of the mutant plasmids, 5× PHUSION® High-FidelityBuffer (New England Biolabs, Inc., Ipswich, Mass., USA), 1 μl of a blendof dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 0.5 μl of PHUSION®High-Fidelity DNA polymerase (New England Biolabs, Inc., Ipswich, Mass.,USA), in a final volume of 50 μl. For pDFng154-17 and the P. emersoniiGH61 polypeptide mutant plasmids, 1.5 μl of DMSO were also added. Theamplification was performed using an EPPENDORF® MASTERCYCLER® 5333programmed for 1 cycle at 98° C. for 30 seconds; 10 cycles each at 98°C. for 10 seconds, 65° C. minus 1° C. per cycle for 30 seconds, and 72°C. for 3 minutes; 25 cycles each at 98° C. for 10 seconds, 55° C. at 30seconds, and 72° C. for 3 minutes; and a final elongation at 72° C. for10 minutes. The heat block then went to a 10° C. soak cycle.

Primer 1201513: (SEQ ID NO: 323) CCAGACCAGCAGAGGAGATAATACTPrimer 1201514: (SEQ ID NO: 324) CAAGGATACCTACAGTTATTCGA

Each PCR reaction was analyzed by 0.7% agarose gel electrophoresis usingTBE buffer where either a 7718 bp PCR product from T. aurantiacus or a7754 bp PCR product band from P. emersonii was observed. The PCRreaction was then digested with 1 μl of Dpn I and 4.5 μl of NEBuffer 4at 37° C. overnight and purified using a QIAGEN® Purification Kitaccording to the manufacturer's instructions.

Example 21: Construction of Aspergillus aculeatus GH61 PolypeptideVariants

The Aspergillus aculeatus GH61 polypeptide variants were constructed bySOE-PCR (Splicing by Overhang Extension Polymerase Chain Reaction) withplasmid pDFng155-33. In brief, the first PCR reaction used forwardprimer BGMH110V2F and a mutation specific reverse primer (Table 13). Thesecond PCR reaction used a reverse primer BGMH109V2R and a mutationspecific forward primer (Table 13) containing the sequence coding forthe altered amino acid. The mutation specific forward and reverseprimers contained 15-20 overlapping nucleotides. The third PCR reactionused the overlapping nucleotides to splice together the fragmentsproduced in the first and second reaction. Finally, using forward primerBGMH110V2F and reverse primer BGMH109V2R, the spliced fragment wasamplified by PCR.

Primer BGMH110V2F: (SEQ ID NO: 325) 5′-CCAGACCAGCAGAGGAGATAATACTCTGCG-3′Primer BGMH109V2R: (SEQ ID NO: 326)5′-CAAGGATACCTACAGTTATTCGAAACCTCCTG-3′

The first SOE-PCR reactions for the A. aculeatus GH61 polypeptidevariants contained 0.5 picomole of the BGMH110V2F primer, 0.5 picomoleof the reverse primer listed in Table 13, 50 ng of template(pDFng155-33), 5 nanomoles each dATP, dTTP, dGTP, and dCTP, PHUSION®High-Fidelity Buffer, and 0.7 unit of PHUSION® High-Fidelity DNAPolymerase, in a final reaction volume of 50 μl. The amplification wasperformed using an EPPENDORF® MASTERCYCLER® Gradient (EppendorfScientific, Inc., Westbury, N.Y., USA) programmed for 1 cycle at 98° C.for 2 minutes; 35 cycles each at 98° C. for 25 seconds, 66° C. for 30seconds, and 72° C. for 5 minute; and a final elongation at 72° C. for10 minutes. The heat block then went to a 10° C. hold stage.

The second SOE-PCR reactions for the A. aculeatus GH61 variantscontained 0.5 picomole of the forward primer listed in Table 13, 0.5picomole of the BGMH109V2R primer, 50 ng of template (pDFng155-33), 5nanomoles each dATP, dTTP, dGTP, and dCTP, PHUSION® High-FidelityBuffer, and 0.7 unit of PHUSION® High-Fidelity DNA Polymerase, in afinal reaction volume of 50 μl. The amplification was performed using anEPPENDORF® MASTERCYCLER® Gradient programmed for 1 cycle at 98° C. for 2minutes; 35 cycles each at 98° C. for 25 seconds, 66° C. for 30 seconds,and 72° C. for 5 minutes; and a final elongation at 72° C. for 10minutes. The heat block then went to a 10° C. hold stage.

Each PCR reaction was analyzed by 1.0% agarose electrophoresis using TAEbuffer where a 3.9 to 6.5 kb (as specified in Table 13) PCR product bandwas observed indicating proper amplification. The remaining 45microliters were then treated with 10 units of Dpn I and 1×NEB4 toremove the remaining wild-type template. The reaction was incubated for1 hour at 37° C. and then purified using a MINELUTE® 96 UF PurificationKit (QIAGEN Inc., Valencia, Calif., USA). The purified PCR products wereresuspended in deionized water to a final volume equal to 20 μl. Theconcentration of each fragment was measured using a NanoDrop 2000(Thermo Scientific, Wilmington, Del., USA).

The third PCR reaction for the A. aculeatus GH61 variants contained 100to 200 ng of each fragment produced in the first and second SOE-PCRreactions, 5 nanomoles each dATP, dTTP, dGTP, and dCTP, 1× PHUSION®High-Fidelity Buffer, and 0.7 units PHUSION® High-Fidelity DNAPolymerase, in a final reaction volume of 50 μl. The amplification wasperformed using an EPPENDORF® MASTERCYCLER® Gradient programmed for 1cycle at 98° C. for 2 minutes; 35 cycles each at 98° C. for 15 seconds,68° C. for 30 seconds, and 72° C. for 10 minutes; and a final elongationat 72° C. for 10 minutes. The heat block then went to a 10° C. holdstage. Primer BGMH110V2F primer (0.5 picomole) and primer BGMH109V2R(0.5 picomole) were added during the annealing/elongation step of thefifth cycle to allow for the overlapping nucleotides to splice.

The wild-type fragment was produced using conditions similar to thethird PCR reaction. The reaction was composed of 50 ng of template(pDFng155-33), 0.5 picomole of primer BGMH110V2F, 0.5 picomole of primerBGMH109V2R, 5 nanomoles each dATP, dTTP, dGTP, and dCTP, 1× PHUSION®High-Fidelity Buffer, and 0.7 units PHUSION® High-Fidelity DNAPolymerase, in a final reaction volume of 50 μl. The amplification wasperformed using an EPPENDORF® MASTERCYCLER® Gradient programmed for 1cycle at 98° C. for 2 minutes; 35 cycles each at 98° C. for 15 seconds,68° C. for 30 seconds, and 72° C. for 10 minutes; and a final elongationat 72° C. for 10 minutes. The heat block then went to a 10° C. holdstage.

Each PCR reaction was analyzed by 1.0% agarose electrophoresis using TAEbuffer where an approximately 8 kb PCR product band was observedindicating proper amplification. The remaining 45 μl of each PCRreaction were then purified using a MINELUTE® 96 UF Purification Kit.The purified PCR products were resuspended in deionized water to a finalvolume equal to 20 μl. The concentration of each fragment was measuredusing a NanoDrop 2000. The entire volume was then transformed into theAspergillus oryzae COLs1300 strain as described in Example 22.

TABLE 13 PCR Amino fragment Template Acid Primer Primer size BackboneSubstitution ID Direction Primer Sequence (kb) Aspergillus D103K 1202768Fwd TCCAGTGGACTACCTGGCCCAAGAGCCACCA 4.1 aculeatusCGGCCCTGTCC (SEQ ID NO: 327) GH61 1202769 RevGGGCCAGGTAGTCCACTGGAGCTCAACAGTA 6.5 C (SEQ ID NO: 328) Aspergillus D103P1202770 Fwd TCCAGTGGACTACCTGGCCCCCCAGCCACCA 4.1 aculeatusCGGCCCTGTCC (SEQ ID NO: 329) GH61 1202769 RevGGGCCAGGTAGTCCACTGGAGCTCAACAGTA 6.5 C (SEQ ID NO: 330) Aspergillus N152I1202771 Fwd CCGGTACCTGGGCCAGTGATATCTTGATCGC 4.0 aculeatusCAACAACAACAGCTG (SEQ ID NO: 331) GH61 1202772 RevATCACTGGCCCAGGTACCGGGGACGTCGTC 6.4 (SEQ ID NO: 332) Aspergillus N152L1202773 Fwd CCGGTACCTGGGCCAGTGATCTCTTGATCGC 4.0 aculeatusCAACAACAACAGCTG (SEQ ID NO: 333) GH61 1202772 RevATCACTGGCCCAGGTACCGGGGACGTCGTC 6.4 (SEQ ID NO: 334) Aspergillus G186F1202774 Fwd AAATCATTGCCCTTCACTCTGCTTTCAACAAG 3.9 aculeatusGATGGTGCTCAGAACTA (SEQ ID NO: 335) GH61 1202775 RevAGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 336) AspergillusG186M 1202776 Fwd AAATCATTGCCCTTCACTCTGCTATGAACAAG 3.9 aculeatusGATGGTGCTCAGAACTA (SEQ ID NO: 337) GH61 1202775 RevAGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 338) AspergillusG186A 1202777 Fwd AAATCATTGCCCTTCACTCTGCTGCCAACAAG 3.9 aculeatusGATGGTGCTCAGAACTA (SEQ ID NO: 339) GH61 1202775 RevAGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 340) AspergillusG186W 1202778 Fwd AAATCATTGCCCTTCACTCTGCTTGGAACAAG 3.9 aculeatusGATGGTGCTCAGAACTA (SEQ ID NO: 341) GH61 1202775 RevAGCAGAGTGAAGGGCAATGATTTCGTGACGG 6.3 AG (SEQ ID NO: 342) AspergillusN187H 1202779 Fwd CATTGCCCTTCACTCTGCTGGTCACAAGGATG 3.9 aculeatusGTGCTCAGAACTACC (SEQ ID NO: 343) GH61 1202780 RevACCAGCAGAGTGAAGGGCAATGATTTCGTGA 6.3 CGG (SEQ ID NO: 344) AspergillusN187K 1202781 Fwd CATTGCCCTTCACTCTGCTGGTAAGAAGGATG 3.9 aculeatusGTGCTCAGAACTACC (SEQ ID NO: 345) GH61 1202780 RevACCAGCAGAGTGAAGGGCAATGATTTCGTGA 6.3 CGG (SEQ ID NO: 346)

Example 22: Expression of the T. aurantiacus GH61A, P. emersonii GH61A,and A. aculeatus GH61 Polypeptides Variants in Aspergillus oryzaeCOLs1300

Aspergillus oryzae COLs1300 was inoculated onto a COVE-N-Gly platecontaining 10 mM urea and incubated at 34° C. until confluent. Sporeswere collected from the plate by washing with 10 ml of YP medium. Thewhole spore suspension was used to inoculate 101 ml of COL1300protoplasting cultivation medium in a 500 ml polycarbonate shake flask.The shake flask was incubated at 30° C. with agitation at 200 rpm for18-24 hours. Mycelia were filtered through a funnel lined withMIRACLOTH® and washed with 200 ml of 0.6 M MgSO₄. Washed mycelia wereresuspended in 10 ml of COLs1300 protoplasting solution in a 125 mlsterile polycarbonate shake flask and incubated at room temperature for3 minutes. One ml of a solution of 12 mg of BSA per ml of deionizedwater was added to the shake flask and the shake flask was thenincubated at 37° C. with mixing at 65 rpm for 45-90 minutes untilprotoplasting was complete. The mycelia/protoplast mixture was filteredthrough a funnel lined with MIRACLOTH® in a 50 ml conical tube andoverlayed with 5 ml of ST. The 50 ml conical tube was centrifuged at1050×g for 15 minutes with slow acceleration/deceleration. Aftercentrifugation, the liquid was separated in 3 phases. The interphasewhich contained the protoplasts was transferred to a new 50 ml conicaltube. Two volumes of STC were added to the protoplasts followed by abrief centrifugation at 1050×g for 5 minutes. The supernatant wasdiscarded and the protoplasts were washed twice with 5 ml of STC withresuspension of the protoplast pellet, centrifugation at 1050×g for 5minutes, and decanting of the supernatant each time. After the finaldecanting, the protoplast pellet was resuspended in STC at aconcentration of 5×10⁷/ml. Protoplasts were frozen at −80° C. untiltransformation.

A 15 μl volume of each mutant fragment, as described in Example 21, wasused to transform 100 μl of A. oryzae COLs1300 protoplasts in a 15 mlround bottom tube. After an initial incubation at room temperature for15 minutes, 300 μl of PEG solution was added to the 15 ml round bottomtube containing the transformation mixture. The reaction was incubatedfor an additional 15 minutes at room temperature. Six ml of melted topagar were added to the reaction and the whole mixture was poured evenlyonto a sucrose agar plate supplemented with 10 mM NaNO₃ and left at roomtemperature until the top agar was set. The plates were incubated at 37°C. for 4-6 days. Resulting transformants were picked using sterileinoculating loops and inoculated into a 96 well flat bottom platecontain 200 μl of MDU2BP per well. The plate was incubated at 34° C.,stationary in a humidified box. Samples were harvested on the third dayby removing the mycelia mat.

Example 23: Determination of Tm (Melting Temperature) of Thermoascusaurantiacus GH61A, Penicillium emersonii GH61A, and Aspergillusaculeatus GH61 Polypeptide Variants by Protein Thermal UnfoldingAnalysis

Protein thermal unfolding of the Thermoascus aurantiacus GH61A,Penicillium emersonii GH61A, and Aspergillus aculeatus GH61 polypeptidevariants was determined by protein thermal unfolding analysis describedaccording to Example 10. The Thermoascus aurantiacus GH61A, Penicilliumemersonii GH61A, and Aspergillus aculeatus GH61 polypeptide variants andwild type polypeptides thereof were prepared as described in Example 22.The results of the thermostability determinations are shown in Table 14.

TABLE 14 Melting temperatures (° C.) of Thermoascus aurantiacus GH61A,Penicillium emersonii GH61A, Aspergillus aculeatus GH61 polypeptidevariants determined by protein thermal unfolding analysis Proteinbackbone Mutations Tm T. aurantiacus GH61 Wild-Type 75 T. aurantiacusGH61 Q188F 78 T. aurantiacus GH61 Q188M 76 P. emersonii GH61 Wild-Type71 P. emersonii GH61 N192M 74 P. emersonii GH61 N193H 73 A. aculeatusGH61 Wild-Type 46 A. aculeatus GH61 D103K 48 A. aculeatus GH61 D103P 48A. aculeatus GH61 N152I 48 A. aculeatus GH61 N152L 49 A. aculeatus GH61G186F 51 A. aculeatus GH61 G186M 51 A. aculeatus GH61 G186A 48 A.aculeatus GH61 G186W 49 A. aculeatus GH61 N187H 48 A. aculeatus GH61N187K 48

The present invention is further described by the following numberedparagraphs:

[1] A GH61 polypeptide variant, comprising a substitution at one or morepositions corresponding to positions 105, 154, 188, 189, 216, and 229 ofthe mature polypeptide of 30, wherein the variant has cellulolyticenhancing activity.[2] The variant of paragraph 1, which has at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the amino acid sequence of a parent GH61 polypeptide.[3] The variant of any of paragraphs 1 or 2, which is a variant of aparent GH61 polypeptide selected from the group consisting of: (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, or 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, or 216; (b) a polypeptide encoded by a polynucleotidethat hybridizes under at least low stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, or 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, or 215, or (ii) the full-length complementof (i); (c) a polypeptide encoded by a polynucleotide having at least60% sequence identity to the mature polypeptide coding sequence of SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131,133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, or 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215;and (d) a fragment of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, or 216, which hascellulolytic enhancing activity.[4] The variant of paragraph 3, wherein the parent GH61 polypeptide hasat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the mature polypeptide of SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.[5] The variant of paragraph 3, wherein the parent GH61 polypeptide isencoded by a polynucleotide that hybridizes under low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, or 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, or 215, or (ii)the full-length complement of (i).[6] The variant of paragraph 3, wherein the parent GH61 polypeptide isencoded by a polynucleotide having at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177, or 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,205, 207, 209, 211, 213, or 215.[7] The variant of paragraph 3, wherein the parent GH61 polypeptidecomprises or consists of the mature polypeptide of SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.[8] The variant of paragraph 3, wherein the parent GH61 polypeptide is afragment of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, or 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, or 215, wherein the fragment hascellulolytic enhancing activity.[9] The variant of any of paragraphs 1-8, which has at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, sequence identity to the mature polypeptide of SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178, or 180, 182, 184, 186, 188, 190, 192, 194,196, 198, 200, 202, 204, 206, 208, 210, 212, 214, or 216.[10] The variant of any of paragraphs 2-9, wherein the variant consistsof at least 85% of the amino acid residues, e.g., at least 90% of theamino acid residues or at least 95% of the amino acid residues of themature polypeptide of the parent GH61 polypeptide.[11] The variant of any of paragraphs 1-10, wherein the number ofsubstitutions is 1-6, e.g., 1, 2, 3, 4, 5, or 6 substitutions.[12] The variant of any of paragraphs 1-11, which comprises asubstitution at a position corresponding to position 105.[13] The variant of paragraph 12, wherein the substitution is Pro orLys.[14] The variant of any of paragraphs 1-13, which comprises asubstitution at a position corresponding to position 154.[15] The variant of paragraph 14, wherein the substitution is Ile orLeu.[16] The variant of any of paragraphs 1-15, which comprises asubstitution at a position corresponding to position 188.[17] The variant of paragraph 16, wherein the substitution is Ala, Met,Phe, or Trp.[18] The variant of any of paragraphs 1-17, which comprises asubstitution at a position corresponding to position 189.[19] The variant of paragraph 18, wherein the substitution is His orLys.[20] The variant of any of paragraphs 1-19, which comprises asubstitution at a position corresponding to position 216.[21] The variant of paragraph 20, wherein the substitution is Leu orTyr.[22] The variant of any of paragraphs 1-21, which comprises asubstitution at a position corresponding to position 229.[23] The variant of paragraph 22, wherein the substitution is Trp, His,Ile, or Tyr.[24] The variant of any of paragraphs 1-23, which comprises asubstitution at two positions corresponding to any of positions 105,154, 188, 189, 216, and 229.[25] The variant of any of paragraphs 1-23, which comprises asubstitution at three positions corresponding to any of positions 105,154, 188, 189, 216, and 229.[26] The variant of any of paragraphs 1-23, which comprises asubstitution at four positions corresponding to any of positions 105,154, 188, 189, 216, and 229.[27] The variant of any of paragraphs 1-23, which comprises asubstitution at five positions corresponding to any of positions 105,154, 188, 189, 216, and 229.[28] The variant of any of paragraphs 1-23, which comprises asubstitution at each position corresponding to positions 105, 154, 188,189, 216, and 229.[29] The variant of any of paragraphs 1-28, which comprises one or moresubstitutions or corresponding substitutions selected from the groupconsisting of E105P,K; E154I,L; G188A,F,M,W; N189H,K; A216L,Y; andK229W,H,I,Y.[30] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K and E154I,L; or corresponding substitutionsthereof.[31] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K and G188A,F,M,W; or corresponding substitutionsthereof.[32] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K and N189H,K; or corresponding substitutionsthereof.[33] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K and A216L,Y; or corresponding substitutionsthereof.[34] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K and

K229W,H,I,Y; or corresponding substitutions thereof.

[35] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L and G188A,F,M,W; or corresponding substitutionsthereof.[36] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L and N189H,K; or corresponding substitutionsthereof.[37] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L and A216L,Y; or corresponding substitutionsthereof.[38] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L and K229W,H,I,Y; or corresponding substitutionsthereof.[39] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W and N189H,K; or corresponding substitutionsthereof.[40] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W and A216L,Y; or corresponding substitutionsthereof.[41] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W and K229W,H,I,Y; or correspondingsubstitutions thereof.[42] The variant of any of paragraphs 1-29, which comprises thesubstitutions N189H,K and A216L,Y; or corresponding substitutionsthereof.[43] The variant of any of paragraphs 1-29, which comprises thesubstitutions N189H,K and K229W,H,I,Y; or corresponding substitutionsthereof.[44] The variant of any of paragraphs 1-29, which comprises thesubstitutions A216L,Y and

K229W,H,I,Y; or corresponding substitutions thereof.

[45] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; and G188A,F,M,W; or correspondingsubstitutions thereof.[46] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; and N189H,K; or correspondingsubstitutions thereof.[47] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; and A216L,Y; or correspondingsubstitutions thereof.[48] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; and K229W,H,I,Y; or correspondingsubstitutions thereof.[49] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; and N189H,K; or correspondingsubstitutions thereof.[50] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; and A216L,Y; or correspondingsubstitutions thereof.[51] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; and K229W,H,I,Y; or correspondingsubstitutions thereof.[52] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; N189H,K; and A216L,Y; or correspondingsubstitutions thereof.[53] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; N189H,K; and K229W,H,I,Y; or correspondingsubstitutions thereof.[54] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; A216L,Y; and K229W,H,I,Y; or correspondingsubstitutions thereof.[55] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; and N189H,K; or correspondingsubstitutions thereof.[56] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; and A216L,Y; or correspondingsubstitutions thereof.[57] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; and K229W,H,I,Y; or correspondingsubstitutions thereof.[58] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; N189H,K; and A216L,Y; or correspondingsubstitutions thereof.[59] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; N189H,K; and K229W,H,I,Y; or correspondingsubstitutions thereof.[60] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; A216L,Y; and K229W,H,I,Y; or correspondingsubstitutions thereof.[61] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W; N189H,K; and A216L,Y; or correspondingsubstitutions thereof.[62] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W; N189H,K; and K229W,H,I,Y; or correspondingsubstitutions thereof.[63] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W; A216L,Y; and K229W,H,I,Y; or correspondingsubstitutions thereof.[64] The variant of any of paragraphs 1-29, which comprises thesubstitutions N189H,K; A216L,Y; and K229W,H,I,Y; or correspondingsubstitutions thereof.[65] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; and N189H,K; orcorresponding substitutions thereof.[66] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; and A216L,Y; orcorresponding substitutions thereof.[67] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; and K229W,H,I,Y; orcorresponding substitutions thereof.[68] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; N189H,K; and A216L,Y; or correspondingsubstitutions thereof.[69] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; N189H,K; and K229W,H,I,Y; orcorresponding substitutions thereof.[70] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[71] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; N189H,K; and A216L,Y; orcorresponding substitutions thereof.[72] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; N189H,K; and K229W,H,I,Y; orcorresponding substitutions thereof.[73] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[74] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; N189H,K; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[75] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; N189H,K; and A216L,Y; orcorresponding substitutions thereof.[76] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; N189H,K; and K229W,H,I,Y; orcorresponding substitutions thereof.[77] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[78] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[79] The variant of any of paragraphs 1-29, which comprises thesubstitutions G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[80] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and A216L,Y; orcorresponding substitutions thereof.[81] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; and K229W,H,I,Y;or corresponding substitutions thereof.[82] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; A216L,Y; and K229W,H,I,Y;or corresponding substitutions thereof.[83] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; N189H,K; A216L,Y; and K229W,H,I,Y; orcorresponding substitutions thereof.[84] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y;or corresponding substitutions thereof.[85] The variant of any of paragraphs 1-29, which comprises thesubstitutions E154I,L; G188A,F,M,W; N189H,K; A216L,Y; and K229W,H,I,Y;or corresponding substitutions thereof.[86] The variant of any of paragraphs 1-29, which comprises thesubstitutions E105P,K; E154I,L; G188A,F,M,W; N189H,K; A216L,Y; andK229W,H,I,Y; or corresponding substitutions thereof.[87] The variant of any of paragraphs 1-86, which further comprises asubstitution at one or more positions corresponding to positions 111,152, 155, and 162 of the mature polypeptide of 30, wherein the varianthas cellulolytic enhancing activity.[88] The variant of paragraph 87, wherein the number of substitutions is1-4, e.g., such as 1, 2, 3, or 4 substitutions.[89] The variant of paragraph 87 or 88, which comprises a substitutionat a position corresponding to position 111.[90] The variant of paragraph 89, wherein the substitution is Val.[91] The variant of any of paragraphs 87-90, which comprises asubstitution at a position corresponding to position 152.[92] The variant of paragraph 91, wherein the substitution is Ser.[93] The variant of any of paragraphs 87-92, which comprises asubstitution at a position corresponding to position 155.[94] The variant of paragraph 93, wherein the substitution is Leu.[95] The variant of any of paragraphs 87-94, which comprises asubstitution at a position corresponding to position 162.[96] The variant of paragraph 95, wherein the substitution is Trp.[97] The variant of any of paragraphs 87-96, which comprises asubstitution at two positions corresponding to any of positions 111,152, 155, and 162.[98] The variant of any of paragraphs 87-96, which comprises asubstitution at three positions corresponding to any of positions 111,152, 155, and 162.[99] The variant of any of paragraphs 87-96, which comprises asubstitution at each position corresponding to positions 111, 152, 155,and 162.[100] The variant of any of paragraphs 87-99, which comprises one ormore substitutions or corresponding substitutions selected from thegroup consisting of L111V, D152S, M155L, and A162W.[101] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+D152S; or corresponding substitutions thereof.[102] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+M155L; or corresponding substitutions thereof.[103] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+A162W; or corresponding substitutions thereof.[104] The variant of any of paragraphs 87-100, which comprises thesubstitutions D152S+M155L; or corresponding substitutions thereof.[105] The variant of any of paragraphs 87-100, which comprises thesubstitutions D152S+A162W; or corresponding substitutions thereof.[106] The variant of any of paragraphs 87-100, which comprises thesubstitutions M155L+A162W; or corresponding substitutions thereof.[107] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+D152S+M155L; or corresponding substitutions thereof.[108] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+D152S+A162W; or corresponding substitutions thereof.[109] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+M155L+A162W; or corresponding substitutions thereof.[110] The variant of any of paragraphs 87-100, which comprises thesubstitutions D152S+M155L+A162W; or corresponding substitutions thereof.[111] The variant of any of paragraphs 87-100, which comprises thesubstitutions L111V+D152S+M155L+A162W; or corresponding substitutionsthereof.[112] The variant of any of paragraphs 1-111, which further comprises asubstitution at one or more positions corresponding to positions 96, 98,200, 202, and 204 of the mature polypeptide of 30, wherein the varianthas cellulolytic enhancing activity.[113] The variant of paragraph 112, wherein the number of substitutionsis 1-5, e.g., such as 1, 2, 3, 4, or 5 substitutions.[114] The variant of paragraph 112 or 113, which comprises asubstitution at a position corresponding to position 96.[115] The variant of paragraph 114, wherein the substitution is Val.[116] The variant of any of paragraphs 112-115, which comprises asubstitution at a position corresponding to position 98.[117] The variant of paragraph 116 wherein the substitution is Leu.[118] The variant of any of paragraphs 112-117, which comprises asubstitution at a position corresponding to position 200.[119] The variant of paragraph 118, wherein the substitution is Ile.[120] The variant of any of paragraphs 112-119, which comprises asubstitution at a position corresponding to position 202.[121] The variant of paragraph 120, wherein the substitution is Leu.[122] The variant of any of paragraphs 112-121, which comprises asubstitution at a position corresponding to position 204.[123] The variant of paragraph 120, wherein the substitution is Val.[124] The variant of any of paragraphs 112-123, which comprises asubstitution at two positions corresponding to any of positions 96, 98,200, 202, and 204.[125] The variant of any of paragraphs 112-123, which comprises asubstitution at three positions corresponding to any of positions 96,98, 200, 202, and 204.[126] The variant of any of paragraphs 112-123, which comprises asubstitution at four positions corresponding to any of positions 96, 98,200, 202, and 204.[127] The variant of any of paragraphs 112-123, which comprises asubstitution at each position corresponding to positions 96, 98, 200,202, and 204.[128] The variant of any of paragraphs 112-127, which comprises one ormore substitutions or corresponding substitutions selected from thegroup consisting of I96V, F98L, F200I, I202L, and I204V.[129] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L; or corresponding substitutions thereof.[130] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F200I; or corresponding substitutions thereof.[131] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+I202L; or corresponding substitutions thereof.[132] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+I204V; or corresponding substitutions thereof.[133] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+F200I; or corresponding substitutions thereof.[134] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+I202L; or corresponding substitutions thereof.[135] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+I204V; or corresponding substitutions thereof.[136] The variant of any of paragraphs 112-128, which comprises thesubstitutions F200I+I202L; or corresponding substitutions thereof.[137] The variant of any of paragraphs 112-128, which comprises thesubstitutions F200I+I204V; or corresponding substitutions thereof.[138] The variant of any of paragraphs 112-128, which comprises thesubstitutions I202L+I204V; or corresponding substitutions thereof.[139] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+F200I; or corresponding substitutions thereof.[140] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+I202L; or corresponding substitutions thereof.[141] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+I204V; or corresponding substitutions thereof.[142] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F200I+I202L; or corresponding substitutions thereof.[143] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F200I+I204V; or corresponding substitutions thereof.[144] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+I202L+I204V; or corresponding substitutions thereof.[145] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+F200I+I202L; or corresponding substitutions thereof.[146] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+F200I+I204V; or corresponding substitutions thereof.[147] The variant of any of paragraphs 112-128, which comprises thesubstitutions F200I+I202L+I204V; or corresponding substitutions thereof.[148] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+I202L+I204V; or corresponding substitutions thereof.[149] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+F200I+I202L; or corresponding substitutionsthereof.[150] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F200I+I202L+I204V; or corresponding substitutionsthereof.[151] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+I202L+I204V; or corresponding substitutionsthereof.[152] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+F200I+I204V; or corresponding substitutionsthereof.[153] The variant of any of paragraphs 112-128, which comprises thesubstitutions F98L+F200I+I202L+I204V; or corresponding substitutionsthereof.[154] The variant of any of paragraphs 112-128, which comprises thesubstitutions I96V+F98L+F200I+I202L+I204V; or correspondingsubstitutions thereof.[155] The variant of any of paragraphs 1-154, wherein thethermostability of the variant is increased at least 1.01-fold, e.g., atleast 1.05-fold, at least 1.1-fold, at least 1.2-fold, at least1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.8-fold, atleast 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, orat least 100-fold compared to the parent.[156] An isolated polynucleotide encoding the variant of any ofparagraphs 1-155.[157] A nucleic acid construct comprising the polynucleotide ofparagraph 156.[158] An expression vector comprising the polynucleotide of paragraph156.[159] A host cell comprising the polynucleotide of paragraph 156.[160] A method of producing a GH61 polypeptide variant, comprising:cultivating the host cell of paragraph 159 under conditions suitable forexpression of the variant.[161] The method of paragraph 160, further comprising recovering thevariant.[162] A transgenic plant, plant part or plant cell transformed with thepolynucleotide of paragraph 156.[163] A method of producing a variant of any of paragraphs 1-155,comprising: cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the variant under conditions conducive forproduction of the variant.[164] The method of paragraph 163, further comprising recovering thevariant.[165] A method for obtaining a GH61 polypeptide variant, comprisingintroducing into a parent GH61 polypeptide a substitution at one or morepositions corresponding to positions 105, 154, 188, 189, 216, and 229 ofthe mature polypeptide of 30, wherein the variant has cellulolyticenhancing activity; and optionally recovering the variant.[166] The method of paragraph 165, further comprising introducing intothe parent GH61 polypeptide a substitution at one or more (e.g.,several) positions corresponding to positions 111, 152, 155, and 162 ofthe mature polypeptide of 30, wherein the variant has cellulolyticenhancing activity.[167] The method of paragraph 165 or 166, further comprising introducinginto the parent GH61 polypeptide a substitution at one or more (e.g.,several) positions corresponding to positions 96, 98, 200, 202, and 204of the mature polypeptide of 30, wherein the variant has cellulolyticenhancing activity.[168] A process for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the GH61 polypeptide variant having cellulolyticenhancing activity of any of paragraphs 1-155.[169] The process of paragraph 168, wherein the cellulosic material ispretreated.[170] The process of paragraph 168 or 169, further comprising recoveringthe degraded cellulosic material.[171] The process of any of paragraphs 168-170, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.[172] The process of paragraph 171, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, aendoglucanase, and a beta-glucosidase.[173] The process of paragraph 171, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.[174] The process of any of paragraphs 168-173, wherein the degradedcellulosic material is a sugar.[175] The process of paragraph 174, wherein the sugar is selected fromthe group consisting of glucose, xylose, mannose, galactose, andarabinose.[176] A process for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the GH61 polypeptide variant having cellulolytic enhancingactivity of any of paragraphs 1-155; (b) fermenting the saccharifiedcellulosic material with one or more fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.[177] The process of paragraph 176, wherein the cellulosic material ispretreated.[178] The process of paragraph 176 or 177, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.[179] The process of paragraph 178, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, aendoglucanase, and a beta-glucosidase.[180] The process of paragraph 178, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.[181] The process of any of paragraphs 176-180, wherein steps (a) and(b) are performed simultaneously in a simultaneous saccharification andfermentation.[182] The process of any of paragraphs 176-181, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.[183] A process of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the GH61 polypeptide varianthaving cellulolytic enhancing activity of any of paragraphs 1-155.[184] The process of paragraph 183, wherein the cellulosic material ispretreated before saccharification.[185] The process of paragraph 183 or 184, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.[186] The process of paragraph 185, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, aendoglucanase, and a beta-glucosidase.[187] The process of paragraph 185, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.[188] The process of any of paragraphs 183-187, wherein the fermentingof the cellulosic material produces a fermentation product.[189] The process of paragraph 189, further comprising recovering thefermentation product from the fermentation.[190] The process of paragraph 188 or 189, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.[191] A whole broth formulation or cell culture composition, comprisingthe variant of any of paragraphs 1-155.[192] A detergent composition, comprising a surfactant and the variantof any of paragraphs 1-155.[193] The composition of paragraph 192, further comprising one or more(e.g., several) enzymes selected from the group consisting of anamylase, arabinase, cutinase, carbohydrase, cellulase, galactanase,laccase, lipase, mannanase, oxidase, pectinase, peroxidase, protease,and xylanase.[194] The composition of paragraph 192 or 193, which is formulated as abar, a tablet, a powder, a granule, a paste, or a liquid.[195] A method for cleaning or washing a hard surface or laundry, themethod comprising contacting the hard surface or the laundry with thecomposition of any of paragraphs 192-194.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1-21. (canceled)
 22. A variant GH61 polypeptide having cellulolyticenhancing activity, comprising a substitution at one or more positionscorresponding to positions 105, 154, 188, 189, 216, and 229 of themature polypeptide of SEQ ID NO: 30, wherein the substitution atcorresponding position 105 is with Pro or Lys, the substitution atcorresponding position 154 is with Ile or Leu, the substitution atcorresponding position 188 is with Ala, Phe, Met, or Trp, thesubstitution at corresponding position 189 is with His or Lys, thesubstitution at corresponding position 216 is with Leu or Tyr, and thesubstitution at corresponding position 229 is with Trp, His, Ile, orTyr, wherein the variant has cellulolytic enhancing activity, whereinthe variant has increased thermostability relative to a GH61 polypeptidewithout the substitution at the one or more positions corresponding topositions 105, 154, 188, 189, 216, and 229 of the mature polypeptide ofSEQ ID NO: 30, and wherein the variant has at least 95% sequenceidentity, but less than 100% sequence identity, to the maturepolypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26,28, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, or
 216. 23. The variant of claim 22, wherein the variant has atleast 96% sequence identity, but less than 100% sequence identity, tothe mature polypeptide of the GH61 polypeptide of SEQ ID NO: 2, 4, 6, 8,10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, or
 216. 24. The variant of claim 22,wherein the variant has at least 97% sequence identity, but less than100% sequence identity, to the mature polypeptide of the GH61polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26,28, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, or
 216. 25. The variant of claim 22, wherein the variant has atleast 98% sequence identity, but less than 100% sequence identity, tothe mature polypeptide of the GH61 polypeptide of SEQ ID NO: 2, 4, 6, 8,10, 12, 16, 18, 20, 22, 24, 26, 28, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, or
 216. 26. The variant of claim 22,wherein the variant has at least 99% sequence identity, but less than100% sequence identity, to the mature polypeptide of the GH61polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 24, 26,28, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, or
 216. 27. The variant of claim 22, which further comprises asubstitution at one or more positions corresponding to positions 111,152, 155, and 162 of the mature polypeptide of SEQ ID NO: 30, whereinthe substitution at corresponding position 111 is with Val, thesubstitution at corresponding position 152 is with Ser, the substitutionat corresponding position 155 is with Leu, and the substitution atcorresponding position 162 is with Trp, and wherein the variant hascellulolytic enhancing activity.
 28. The variant of claim 27, whereinsaid variant comprises one or more substitutions or correspondingsubstitutions selected from the group consisting of a substitution ofLeu at position 111 with Val, a substitution of Asp at position 152 withSer, a substitution of Met at position 155 with Leu, and a substitutionof Ala at position 162 with Trp.
 29. The variant of claim 22, whereinthe thermostability of the variant is increased at least 1.01-foldcompared to the parent.
 30. An isolated polynucleotide encoding the GH61polypeptide variant of claim
 22. 31. A recombinant host cell transformedwith the polynucleotide of claim
 30. 32. A method of producing a GH61polypeptide variant, comprising: (a) cultivating the recombinant hostcell of claim 31 under conditions suitable for expression of thevariant; and optionally (b) recovering the variant.
 33. A transgenicplant, plant part or plant cell transformed with the polynucleotide ofclaim
 30. 34. A method of producing a GH61 polypeptide variant,comprising: (a) cultivating the transgenic plant or a plant cell ofclaim 33 under conditions conducive for production of the variant; andoptionally (b) recovering the variant.
 35. A process for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition comprising the GH61 polypeptidevariant having cellulolytic enhancing activity of claim
 22. 36. Aprocess for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme compositioncomprising the GH61 polypeptide variant having cellulolytic enhancingactivity of claim 22; (b) fermenting the saccharified cellulosicmaterial with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.
 37. A process of fermenting a cellulosic material,comprising: fermenting the cellulosic material with one or morefermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition comprising the GH61 polypeptidevariant having cellulolytic enhancing activity of claim
 22. 38. A wholebroth formulation or cell culture composition, comprising the GH61polypeptide variant of claim
 22. 39. A detergent composition, comprisinga surfactant and the GH61 polypeptide variant of claim 22.